BIOLOGY OF REPRODUCTION 63, 1913–1917 (2000)
Apoptosis in Rat Placenta Is Zone-Dependent and Stimulated by Glucocorticoids1
Brendan J. Waddell,2 Susan Hisheh, A.M. Dharmarajan, and Peter J. Burton
Department of Anatomy & Human Biology, The University of Western Australia, Nedlands, Western Australia
ABSTRACT
Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor
in the overall control of feto-placental growth. The present study
used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones,
during the last week of pregnancy (Days 16, 22, and 23). In
addition, because glucocorticoids are potent inhibitors of fetoplacental growth and can stimulate apoptosis in other tissues,
we examined whether dexamethasone treatment in vivo induces
placental apoptosis. DNA fragmentation was clearly evident in
both placental zones at each stage of pregnancy, with higher
levels evident in the basal zone compared with the labyrinth
zone on Days 22 and 23. TUNEL analysis, which identifies dying
cells in situ, demonstrated positive staining of cells in the basal
zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not
the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11b-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone
but not in the labyrinth zone. These effects of dexamethasone
and carbenoxolone on placental apoptosis were associated with
reduced placental and fetal weights. In conclusion, this study
shows that apoptosis occurs in both zones of the rat placenta,
particularly in the basal zone near term, and is elevated after
increased glucocorticoid exposure in vivo. These data support
the hypothesis that placental apoptosis is an important player in
the regulation of feto-placental growth, and establish the rat as
a useful model to study the endocrine control of placental apoptosis.
apoptosis, conceptus, corticosterone, placenta, pregnancy
INTRODUCTION
Placental growth, development, and aging are crucial to
the overall well-being of the fetus and are controlled by a
range of endocrine signals, including steroids and growth
factors [1, 2]. Apoptosis, or physiological cell death, has
been observed in the human placenta, particularly in the
latter half of pregnancy [3] when its incidence increases
along with the expression of T-18, a recently identified,
apoptosis-associated gene [4]. Placental apoptosis also increases in association with fetal growth retardation [5]. In
other tissues, apoptosis is recognized as a key player in
normal development and tissue homeostasis, facilitating
rapid removal of unwanted cells in the absence of inflamSupported by the National Health and Medical Research Council of Australia (project grant 970132).
Correspondence. FAX: 61 8 9380 1051;
e-mail: [email protected]
1
2
Received: 20 December 1999.
First decision: 18 January 2000.
Accepted: 25 July 2000.
Q 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
mation [6]. Thus, the association between placental apoptosis and fetal growth retardation [5] suggests that the endocrine control of feto-placental growth may involve a balance between trophic and apoptotic signals within the placenta. It has also been postulated that placental apoptosis
may affect a range of placental functions [7], including placental remodeling to control the syncytiotrophoblast:cytotrophoblast ratio [8]. Given these potentially crucial roles
of apoptosis in the placenta, the present study used DNA
internucleosomal fragmentation analysis to characterize
placental apoptosis over the final third of rat pregnancy, the
period of maximal fetal growth. The two morphologically
and functionally distinct regions of the rat placenta, the
basal and labyrinth zones, were analyzed separately. The
basal zone is the major site of placental hormone production over late pregnancy [9], whereas the labyrinth zone is
the major site of feto-maternal exchange [10]. These initial
analyses showed clear evidence of zone- and stage-specific
differences in placental apoptosis. To investigate potential
endocrine regulation of this dynamic apoptotic pattern, placental apoptosis was measured after maternal treatment
with glucocorticoids in vivo, which are known to stimulate
apoptosis in other tissues [11] and are potent inhibitors of
feto-placental growth [12–14]. Glucocorticoid treatment
clearly stimulated placental apoptosis, and so a final experiment examined the effects of carbenoxolone, an inhibitor of 11b-hydroxysteroid dehydrogenase (11b-HSD). This
enzyme controls access of endogenous glucocorticoids to
their receptors in target tissues (for a review, see [15]),
including the placenta (for a review, see [16]).
MATERIALS AND METHODS
Animals, Treatments, and Tissue Collection
Nulliparous albino Wistar rats, 3–5 mo old (Animal Resources Centre, Murdoch Australia), were used in this
study. Rats were mated overnight and the morning on
which spermatozoa were present in a vaginal smear was
designated as Day 1 of pregnancy. All procedures involving
animals were conducted only after approval by the Animal
Experimentation Ethics Committee of The University of
Western Australia. Placentas were collected from rats anesthetized with halothane/nitrous oxide on Days 16, 22, and
23 of pregnancy (term 5 Day 23), dissected on ice into
basal and labyrinth zones [17], weighed to the nearest milligram, frozen on liquid nitrogen, and stored at 2808C.
Dexamethasone acetate (Sigma Chemical Co, St. Louis,
MO) was administered via drinking water at two doses
(0.25 and 1 mg/ml) from Day 15 to 22 of pregnancy; similar
treatments have recently been shown to reduce birth weight
by 9% and 27%, respectively [18]. Carbenoxolone (Sigma)
was administered as twice daily s.c. injections (10 mg in
0.1 ml 4% ethanol in saline) from Day 13 to Day 22 of
pregnancy; this treatment has been shown to reduce birth
weight in the rat by 8% [18]. Sham control rats were injected twice daily with 0.1 ml 4% ethanol in saline. Placental zones were collected from treatment groups on Day
22 of pregnancy and processed as just described.
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and a series of phenol/chloroform extractions, and the purity and concentration of extracted DNA was assessed by
spectrophotometry. The extent of DNA internucleosomal
fragmentation was determined by end-labeling using terminal transferase to bind radioactively labeled ddATP to 39
ends of double- and single-stranded DNA [20]. The reaction was composed of the following: 1 mg DNA made up
to 29 ml volume with dH2O, 10 ml 53 reaction buffer (1
M potassium cacodylate, 125 mM Tris-HCl, 1.25 mg/ml
BSA pH 6.6), 5 ml 25 mM CoCl2, 1 ml terminal transferase
(diluted to 2.5 U/ml with dH2O; Boehringer, Mannheim,
Germany), and 5 ml [a32P]ddATP (17 pmol; 50 mCi; Amersham, Australia). The reaction was incubated for 60 min at
378C and terminated by 5 ml 0.25 M EDTA followed by
the addition of 2 ml tRNA (Boehringer). DNA was then
precipitated using 10 M ammonium acetate and ethanol and
incubated at 2808C for 60 min. The DNA pellet was collected, resuspended in 13 TE pH 8.0, and precipitated
again as described earlier. The resulting pellet was air-dried
and resuspended in TE and stored at 2208C until electrophoresis. DNA fragments were separated on a 2% agarose
gel in 13 TAE buffer for 3 h at 60 V. The gel was then
dried in a slab-gel dryer for 2 h before being exposed to
Kodak X-Omat film (Kodak Australia, Coburg, Australia)
overnight at 2808C. The extent of internucleosomal DNA
fragmentation was observed by the incorporation of [a32P]ddATP onto the 39 ends of low molecular weight (,20
kb) fragments. These low molecular weight bands were excised (starting 2.5 cm from the origin but discarding the
high molecular weight DNA) and quantified by measuring
emissions in a liquid scintillation spectrometer.
TUNEL Analysis
Specific placental cells undergoing cell death were identified by TUNEL analysis using a kit obtained from Introgen Co. (Purchase, NY). Briefly, placentas were immersefixed in 4% paraformaldehyde, processed for routine paraffin histology; sections (5 mm) were deparaffinized, pretreated with proteinase K (20 mg/ml), quenched with 3%
hydrogen peroxide, and the TUNEL procedures conducted
according to maufacturer’s instructions. TUNEL-positive
cells were visualized with diaminobenzidine and counterstained with methyl green.
Statistical Analysis
FIG. 1. Low molecular weight DNA fragmentation in placental zones
(B, basal; L, labyrinth) at Days 16, 22, and 23 of pregnancy (term 5 Day
23). Top) Representative autoradiograph of placental DNA fragmentation.
Bottom) Quantitation of DNA fragmentation in placental zones. Values
are the mean 6 SEM (n 5 3 per group). Asterisk indicates significant
difference between zones P , 0.05 (paired t-test).
A maximum of one fetus/placenta was obtained from
any given mother, such that references to ‘n’ are for the
number of mothers in each group. The extent of DNA fragmentation in the two placental zones over the 3 days of
pregnancy was assessed by two-way ANOVA and least significant difference (LSD) tests; differences between placental zones on each day were also assessed by paired t tests
[21]. Changes in placental zone weights and fetal weight
were determined by a one-way ANOVA within each category (basal, labyrinth, fetus) and LSD tests [21]. The effect
of dexamethasone treatments on DNA fragmentation was
assessed by one-way ANOVA and LSD tests, and the effect
of carbenoxolone by unpaired t tests.
RESULTS
Analysis of Internucleosomal DNA Fragmentation
Effect of Gestational Age on Placental Apoptosis
DNA was isolated from placental zones by the method
described by Dharmarajan et al. [19]. Tissue was homogenized, protein was denatured and then removed with salts
DNA fragmentation that was indicative of apoptosis was
clearly present in the basal and labyrinth zones of the placenta at each stage of pregnancy examined (Fig. 1), and
APOPTOSIS IN RAT PLACENTA
FIG. 2. In situ placental cell death demonstrated by TUNEL analysis at
Day 22 of pregnancy. Positive TUNEL staining was readily apparent in
the basal zone. Note positive staining of a giant trophoblast cell (GTC),
and absence of staining in neighboring cells (3400).
there was a highly significant effect of placental zone evident (P , 0.001, two-way ANOVA). There was also significant interaction between the day of pregnancy and placental zone (P 5 0.05), indicating that the difference in
apoptosis between basal and labyrinth zones was stage-dependent. Accordingly, whereas comparable levels of apoptosis were evident in the two zones on Day 16, DNA
fragmentation in the basal zone clearly exceeded (P , 0.05)
that in the labyrinth zone on Day 22, and this difference
was at least maintained to Day 23 (P , 0.01; Fig. 1).
TUNEL analysis clearly demonstrated death of giant trophoblast cells in the basal zone (Fig. 2).
Effect of Dexamethasone and Carbenoxolone
on Placental Apoptosis
Dexamethasone treatment at the higher of the two doses
(1 mg/ml dexamethasone acetate in drinking water) increased (72%; P , 0.05) DNA fragmentation in the basal
zone but not the labyrinth zone (Fig. 3). Similarly, maternal
treatment with carbenoxolone, an inhibitor of 11b-HSD,
FIG. 3. Effect of dexamethasone on placental DNA fragmentation in placental zones at Day 22 of pregnancy. Values are the mean 6 SEM (n 5
4–5 per group). Those values without shared notations differ at P , 0.05
(one-way ANOVA and LSD test).
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FIG. 4. Effect of carbenoxolone (CBX) treatment on DNA fragmentation
in placental zones at Day 22 of pregnancy. Values are the mean 6 SEM.
DNA fragmentation for each placental zone did not differ significantly
between vehicle-treated and untreated rats, and so these values were
combined (control; n 5 12) for comparison with the CBX group (n 5 5).
* P , 0.05 compared with control (unpaired t-test).
increased DNA fragmentation in the basal zone (fourfold;
P , 0.05) but had no effect in the labyrinth zone (Fig. 4).
Effect of Dexamethasone and Carbenoxolone on Fetal
and Placental Weight
Dexamethasone treatment reduced (P , 0.05) the weight
of the basal zone at both doses tested (32% and 46% decrease after low- and high-dose treatments, respectively),
and similar effects were evident for labyrinth-zone weight
(15% and 28% lower, respectively; P , 0.01; Fig 5). Fetal
weight was unaffected by the lower-dose dexamethasone
treatment but was clearly reduced after higher-dose dexamethasone (29% reduction, P , 0.01). Carbenoxolone
treatment also reduced labyrinth and fetal weights (16%
FIG. 5. Fetal and placental zone weights following dexamethasone
(DEX) and carbenoxolone (CBX) treatments. Values are mean 6 SEM (n
5 4–5 per group). Those values without shared notations differ at P ,
0.05 (one-way ANOVA and LSD test).
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WADDELL ET AL.
and 12% lower, respectively; P , 0.05), and although basal
zone weight appeared to fall slightly, this did not reach
statistical significance (Fig. 5).
DISCUSSION
Adequate placental growth and function are fundamental
to the well-being, growth, and development of the fetus
throughout gestation. A complex interplay among fetal, placental, and maternal hormones regulates the rate of placental growth [1, 2], and recent studies have identified apoptosis, or physiological cell death, as a potentially important
determinant of placental size and function. Thus, Smith et
al. [5] demonstrated that placental apoptosis is elevated in
human pregnancies complicated by intrauterine growth retardation. Placental apoptosis also increases over the course
of normal human pregnancy [3], but the factors regulating
this elevation and its functional implications for normal placental growth are unknown. Development of an animal
model to study the link between feto-placental growth and
placental apoptosis is thus important, and so the present
study investigated the extent of apoptosis in the rat placenta
over the final third of pregnancy, the period of maximal
fetal growth. The rat placenta is a particularly useful model
because it is composed of two morphologically and functionally distinct regions, the basal and labyrinth zones. The
basal zone consists of trophoblast and maternal vessels but
no fetal vessels [10], and is the major site of hormone production [9]. The labyrinth zone consists of trophoblast cells
and maternal and fetal vessels, and is thus the major site
of feto-maternal exchange [10]. The major findings of the
present work were that apoptosis was clearly evident in
both placental zones, and was particularly prevalent in the
basal zone near term. TUNEL analysis showed that giant
trophoblast cells accounted for most of the cell death in this
zone. We also demonstrated that basal zone apoptosis was
stimulated by increased glucocorticoid exposure, which is
well-recognized as a potent inhibitor of fetal growth. Finally, inhibition of 11b-HSD by treatment with carbenoxolone also stimulated placental apoptosis and reduced fetal
growth, which is suggestive of a protective role for placental 11b-HSD with respect to endogenous glucocorticoids.
The clearly higher level of apoptosis in the basal zone
compared with the labyrinth zone at term is consistent with
the higher growth rate of the labyrinth zone during late
pregnancy. Thus, while basal zone weight remains unchanged from Day 16 to Day 22 of pregnancy, the labyrinth
zone, where we observed relatively little apoptosis, increases in weight more than threefold over this period of high
fetal demand [22], which is consistent with its role in fetomaternal exchange [10]. The higher rate of apoptosis in the
basal zone may reflect reduced functional importance of
this tissue near term; indeed, basal zone synthesis of androgens and progesterone falls precipitously in late pregnancy [9].
The stimulation of placental apoptosis by glucocorticoids is consistent with the known effect of these steroids
on fetal growth. Thus, maternal administration of synthetic
glucocorticoids has been shown to reduce fetal growth in
several species [12–14, 23] and this is associated with reduced placental growth [13, 14]. Glucocorticoids are known
to be proapoptotic in a number of tissues [11], although it
is interesting that they can be antiapoptotic in some cell
types [24]. It is not yet clear whether the effects of dexamethasone on placental apoptosis (and the comparable effects of carbenoxolone, see later discussion) are due to direct, glucocorticoid stimulation of proapoptotic genes with-
in placental cells, or are secondary to compromised fetoplacental function. For example, glucocorticoids are
well-recognized as inhibitors of the inducible form of nitric
oxide synthase (iNOS) [25–27], and Miller et al. [28] recently demonstrated that iNOS inhibition in pregnant rats
leads to increased placental cell death, possibly reflecting
apoptosis. Moreover, glucocorticoids can reduce placental
progesterone synthesis [29], and progesterone has been
shown to exert an antiapoptotic effect in rat decidua [30].
Clearly, further studies are required to elucidate these possible mechanisms of glucocorticoid-induced placental apoptosis.
The relative effects of low-dose dexamethasone treatment on growth of the placenta and fetus may provide some
insight into the site of glucocorticoid action. Thus, weights
of both basal and labyrinth placental zones were reduced
after administration of dexamethasone acetate at a dose of
0.25 mg/ml in drinking water (by 32% and 15%, respectively), but fetal weight was unaffected. Only after the higher-dose dexamethasone treatment was fetal growth compromised, raising the possibility of a threshold effect of dexamethasone on fetal growth. Moreover, these observations
suggest that the placenta may be more susceptible than the
fetus to glucocorticoid-induced growth inhibition, with likely flow-on effects to fetal well-being and growth. This contention is supported by recent reports showing that maternally administered betamethasone promotes fetal lung differentiation and reduces fetal and placental growth, whereas
fetally administered betamethasone promotes lung development but is without effect on fetal growth [14, 23]. Because the concentration of betamethasone within the fetal
compartment was comparable in these two treatment regimens, it appears that maternally administered betamethasone may retard fetal growth via a placental mechanism
[14, 23]. This suggestion is supported by our observation
that low-dose dexamethasone treatment reduced placental
zone weights but not fetal weight. Our data further suggest
that if such a placentally mediated effect of glucocorticoids
is operable, it may involve increased placental apoptosis.
The effects of carbenoxolone on placental apoptosis support an important role for endogenous glucocorticoids in
the normal control of feto-placental growth. Carbenoxolone
inhibits the enzyme 11b-HSD, which in placenta constitutes
the ‘‘placental glucocorticoid barrier’’ and is also likely to
control access of active glucocorticoid to its receptor in
placental cells [16]. By inhibiting this enzyme, the placenta
and, thus, the fetus are exposed to higher levels of endogenous glucocorticoid [31], and so theoretically, carbenoxolone should mimic the effects of exogenous glucocorticoids such as dexamethasone, because the latter is a relatively poor substrate for 11b-HSD. We have previously
shown that by the end of rat pregnancy, only the basal zone
of the placenta expresses significant amounts of the 11bHSD-2 isoform, with levels in the labyrinth zone falling
dramatically between Days 16 and 22 of pregnancy [22,
32]. Because this 11b-HSD-2 enzyme acts as an 11b-dehydrogenase to inactivate corticosterone (the major active
glucocorticoid of the rat), its inhibition by carbenoxolone
at term would be expected to increase glucocorticoid exposure in the basal zone but have little effect in the labyrinth zone. Our observation that carbenoxolone treatment
stimulated apoptosis in basal zone but not the labyrinth
zone is consistent with this pattern of 11b-HSD-2 expression. It is interesting that this effect of carbenoxolone in
the basal zone was not accompanied by a significant effect
on basal zone weight. This presumably reflects the rela-
APOPTOSIS IN RAT PLACENTA
tively low expression of 11b-HSD-2 in the basal zone until
late in pregnancy (.threefold increase from Day 16 to Day
22) [22, 32], which should render this zone resistant to
carbenoxolone effects until just prior to term. It is also noteworthy in this context that although DNA fragmentation
provides evidence of recent apoptosis (because apoptotic
cells are normally removed within hours [6]), tissue weight
changes more likely reflect treatment effects over the entire
treatment period (in this case 7 days). Overall, therefore,
the effects of carbenoxolone on basal zone apoptosis and
weight suggest that in normal pregnancy, 11b-HSD-2 expression reduces levels of active, endogenous glucocorticoids in this zone near term and thereby limits their induction of apoptosis. Such a role for placental 11b-HSD in the
control of endogenous glucocorticoid levels within the fetoplacental compartment is also supported by the observation
that birth weight is positively associated with placental
11b-HSD bioactivity at term [33].
In conclusion, this study shows that placental apoptosis
is characteristic of normal rat pregnancy and occurs in a
zone- and time-specific manner. Placental apoptosis was
clearly stimulated by exogenous glucocorticoids and by the
11b-HSD inhibitor, carbenoxolone, an effect that is likely
mediated via increased placental exposure to endogenous
glucocorticoids. Collectively, these data suggest that placental apoptosis may be a key mechanism that is operable
in the complex control of feto-placental growth.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
ACKNOWLEDGMENTS
23.
We gratefully acknowledge the assistance of Melissa Berg with the
TUNEL analysis and the technical assistance of Steve Parkinson.
24.
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