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a 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 feto-placental 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 11ß-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 inflammation [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 11ß-hydroxysteroid dehydrogenase (11ß-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 = Day 23), dissected on ice into basal and labyrinth zones [17], weighed to the nearest milligram, frozen on liquid nitrogen, and stored at -80°C. Dexamethasone acetate (Sigma Chemical Co, St. Louis, MO) was administered via drinking water at two doses (0.25 and 1 µg/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.
Analysis of Internucleosomal DNA Fragmentation
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 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 3' ends of double- and single-stranded DNA [20]. The reaction was composed of the following: 1 µg DNA made up to 29 µl volume with dH2O, 10 µl 5x reaction buffer (1 M potassium cacodylate, 125 mM Tris-HCl, 1.25 mg/ml BSA pH 6.6), 5 µl 25 mM CoCl2, 1 µl terminal transferase (diluted to 2.5 U/µl with dH2O; Boehringer, Mannheim, Germany), and 5 µl [
32P]ddATP (17 pmol; 50 µCi; Amersham, Australia). The reaction was incubated for 60 min at 37°C and terminated by 5 µl 0.25 M EDTA followed by the addition of 2 µl tRNA (Boehringer). DNA was then precipitated using 10 M ammonium acetate and ethanol and incubated at -80°C for 60 min. The DNA pellet was collected, resuspended in 1x TE pH 8.0, and precipitated again as described earlier. The resulting pellet was air-dried and resuspended in TE and stored at -20°C until electrophoresis. DNA fragments were separated on a 2% agarose gel in 1x 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 -80°C. The extent of internucleosomal DNA fragmentation was observed by the incorporation of [-32P]ddATP onto the 3' 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 immerse-fixed in 4% paraformaldehyde, processed for routine paraffin histology; sections (5 µm) were deparaffinized, pretreated with proteinase K (20 µg/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
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
Effect of Gestational Age on Placental Apoptosis
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 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 = 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).
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Effect of Dexamethasone and Carbenoxolone on Placental Apoptosis
Dexamethasone treatment at the higher of the two doses (1 µg/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 11ß-HSD, increased DNA fragmentation in the basal zone (fourfold; P < 0.05) but had no effect in the labyrinth zone (Fig. 4).
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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% and 12% lower, respectively; P < 0.05), and although basal zone weight appeared to fall slightly, this did not reach statistical significance (Fig. 5).
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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 11ß-HSD by treatment with carbenoxolone also stimulated placental apoptosis and reduced fetal growth, which is suggestive of a protective role for placental 11ß-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 feto-maternal 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 within placental cells, or are secondary to compromised feto-placental 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 µg/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 11ß-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 11ß-HSD. We have previously shown that by the end of rat pregnancy, only the basal zone of the placenta expresses significant amounts of the 11ß-HSD-2 isoform, with levels in the labyrinth zone falling dramatically between Days 16 and 22 of pregnancy [22, 32]. Because this 11ß-HSD-2 enzyme acts as an 11ß-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 11ß-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 relatively low expression of 11ß-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, 11ß-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 11ß-HSD in the control of endogenous glucocorticoid levels within the feto-placental compartment is also supported by the observation that birth weight is positively associated with placental 11ß-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 11ß-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.
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance of Melissa Berg with the TUNEL analysis and the technical assistance of Steve Parkinson.
FOOTNOTES
First decision: 18 January 2000.
1 Supported by the National Health and Medical Research Council of Australia (project grant 970132). 
2 Correspondence. FAX: 61 8 9380 1051; bwaddell{at}anhb.uwa.edu.au
Accepted: July 25, 2000.
Received: December 20, 1999.
REFERENCES
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