Tài liệu Báo cáo Y học: Progressive apoptosis in chorion laeve trophoblast cells of human fetal

Progressive apoptosis in chorion laeve trophoblast cells of human fetal

membrane tissues during in vitro incubation is suppressed by

antioxidative reagents

Kunio Ohyama1

, Bo Yuan1

, Toshio Bessho2 and Toshio Yamakawa1

1

Department of Biochemistry, Faculty of Pharmacy, Tokyo University of Pharmacy & Life Science, Tokyo, Japan; 2

Yoneyama Maternity

Hospital, Tokyo, Japan

Previously, we demonstrated apoptotic cell death in the

chorion laeve trophoblast layer of human fetal membrane

tissues during the late stages of pregnancy, the progression

of apoptosis during incubation in vitro, and its suppression

by a low concentration of glucocorticoid hormones. We now

report examination of mRNA expression of inflammatory

cytokines [interleukin (IL)-1b, IL-6, tumor necrosis factor￾a] and antioxidative enzyme genes [heme oxygenase 1,

catalase, Mn-superoxide dismutase (SOD), Cu/Zn-SOD,

glutathione S-transferase, glutathione reductase and gluta￾thione peroxidase] and apoptosis-related genes during in

vitro progression of apoptosis with or without glucocorti￾coid by a reverse transcription/PCR method. It was shown

that the mRNA levels increased in chorion laeve tissue for

each cytokine examined and for catalase, heme oxygenase 1

and Mn-SOD in direct correlation with the in vitro

incubation period. By Western blotting the existence of

Mn-SOD protein, and its slight increase with incubation

time, was also shown. The investigation of the influence of

antioxidative reagents [pyrrolidine dithiocarbamate

(PDTC), N-acetyl-L-cysteine (NAC) and nordihydro￾guaiaretic acid (NDGA)] on DNA fragmentation showed

that DNA fragmentation in chorion laeve tissues was

inhibited by < 50% in the presence of 1 mM PDTC, 30 mM

NAC and 1 mM NDGA. These results suggest that apoptotic

cell death of the trophoblast layer of chorion tissues may be

induced through intracellular oxidative stress at the stage of

parturition.

Keywords: antioxidant; apoptosis; chorion; fetal membrane;

oxidative stress.

We have previously reported that (a) a substantial population

of trophoblast cells in the chorion laeve tissue of human fetal

membrane are induced to undergo apoptosis at the end of

pregnancy, (b) that apoptosis progresses rapidly in vitro, and

(c) that apoptosis of the trophoblasts in the tissue was

suppressed by the presence of a low concentration of

glucocorticoids, hydrocortisone and cortisone [1]. Further￾more, we found that cultivated trophoblasts prepared

primarily from the fetal membrane were induced to undergo

apoptosis by tumor necrosis factor (TNF)-a and also that

apoptosis was inhibited by low concentrations of gluco￾corticoids [1]. From these results we suggested that

apoptotic death of trophoblasts plays an important role in

spontaneous disruption of the fetal membrane tissues during

the final stage of gestation, and that any inflammatory and/or

oxidative events may have a central role in the induction of

trophoblast apoptosis.

Pregnancy is a physiological state accompanied by an

increased requirement for tissue oxygen [2]. It was shown by

in vitro investigation that the increased oxygen requirement

during embryo development increases the rate of reactive

oxygen species (ROS) production [3]. It has also been

established that ROS damage cell membrane lipids and

induce lipid peroxide formation [4,5] as circulating markers

of oxidative stress are increased during normal pregnancy.

These peroxidized lipids are produced mainly in the

placenta [6] and increase in the blood of pregnant women

[7]. These observations indicate that there is an increased

oxidative stress during pregnancy. It is common knowledge

in the field of obstetrics and gynecology that the beginning

of spontaneous disruption of fetal membrane may be caused

by tissue inflammation. Furthermore, injury caused by

oxidative stress may be responsible for developmental

retardation and arrest of mammalian preimplantation

embryos in vitro [8,9]. However, no detail is known about

the mechanism of the disruption and its relationship to the

induction of apoptosis.

Our hypothesis is that oxidative stress contributes to ROS

formation in the placenta and increases gradually during

pregnancy. The stress may serve as a signal to initiate and

propagate the inflammatory process and result in apoptosis

of placental tissues. Accordingly, we investigated the

correlation between the progression of trophoblast

apoptosis and the induction of inflammatory cytokine and/

or cellular oxidative stress occurrence. In this paper, we

describe trophoblast cell apoptosis during in vitro

incubation of human fetal membrane tissue, transcription

of apoptosis-related genes, inhibition of apoptosis by

Correspondence to K. Ohyama, Department of Biochemistry, School of

Pharmacy, Tokyo University of Pharmacy & Life Science, 1432-1

Horinouchi, Hachioji, Tokyo 192-03, Japan. Fax: 181 426 76 5736,

Tel.:181 426 76 5792, E-mail: [email protected]

(Received 20 June 2001, revised 2 October 2001, accepted 4 October

2001)

Abbreviations: TNF, tumor necrosis factor; ROS, reactive oxygen

species; PDTC, pyrrolidine dithiocarbamate; NAC, N-acetyl￾L-cysteine; NDGA, nordihydroguaiaretic acid; IL, interleukin; NFkB,

nuclear factor kb; TNFR, TNF receptor; SOD, superoxide dismutase.

Eur. J. Biochem. 268, 6182–6189 (2001) q FEBS 2001