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Department of Medicine (B.J.W., R.B., R.W.B., J.R.S.), The University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU Scotland, United Kingdom; and Department of Anatomy and Human Biology (B.J.W.), The University of Western Australia, Nedlands, Perth, Western Australia 6907, Australia
Address all correspondence and requests for reprints to: Dr. Brendan J. Waddell, Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, Western Australia 6907, Australia. E-mail: bwaddell{at}anhb.uwa.edu.au
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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In situ hybridization
Rat complementary DNA (cDNA) clones for 11ß-HSD-1 (12),
11ß-HSD-2 (13), the GR (14), and MR (15) were linearized with
appropriate restriction enzymes (Promega Ltd., Southampton, UK), and
the resultant templates used with 35S-
-UTP (>1000
Ci/mmol; Amersham, Aylesbury, UK) to synthesize antisense and sense
cRNA probes as previously described (16). Cryostat sections (15 µm)
of whole placenta were thaw-mounted onto gelatin- and
poly-L-lysine-coated microscope slides and stored at -80
C. Tissues sections were postfixed in 4% paraformaldehyde/phosphate
(0.1 mol/liter) buffer and prehybridized, hybridized, and washed as
previously described (16, 17). Following hybridization and washing, all
slides were placed against hyperfilm ßmax to detect
positive signals for mRNAs; selected slides were subsequently dipped in
photographic emulsion (NTB2, Eastman Kodak Company, Rochester, NY) to
allow cellular localization of these signals which also required
counterstaining with hematoxylin and eosin.
11ß-HSD bioassays
Placentas from all untreated rats, adrenalectomized rats, and
from the untreated horn of fetectomized rats were separated into basal
and labyrinth zones as described by Chan and Leatham (18); fetectomized
placentas could not be reliably separated into these zones so were left
intact. Tissues were then placed in ice-cold PBS (pH 7.4) containing
0.25 M sucrose and homogenized. The homogenate was
centrifuged at 750 x g for 10 min at 4 C, and the
protein concentration of the supernatant determined (Bio-Rad protein
assay kit, Bio-Rad, Hertfordshire, UK). 11ß-HSD activity was
determined by measuring the conversion of
[1,2,6,7-3H]corticosterone (SA 90 Ci/mmol; Amersham) (10
nM) to [3H]11-dehydrocorticosterone by 500
µg/ml supernatant protein in the presence of 400 µM
NADP (for 11ß-HSD-1 activity) or NAD+ (for 11ß-HSD-2
activity) and varying concentrations of added cold corticosterone (0.5,
1, 2, 5, 10, 20 µM for 11ß-HSD-1; 0, 12.5, 25, 50, 100,
200 nM for 11ß-HSD-2) (16). At each concentration,
duplicate incubations were performed at 37 C for 10 min then stopped by
the addition of 2.5 ml ethyl acetate, into which steroids were
extracted. Extracts were dried and [3H]corticosterone and
[3H]11-dehydrocorticosterone purified and quantitated by
HPLC as previously described (19). Preliminary experiments established
that conversion of [3H]corticosterone to
[3H]11-dehydrocorticosterone remained linear beyond the
10-min incubation time under these assay conditions. The reaction
velocity (pmol/min per mg protein) for each duplicate incubation set
was used to construct Lineweaver-Burk plots for individual
placental tissues, from which each Km and
Vmax were derived (20). For each animal (or
treatment within animals), tissues from two placentas were analyzed,
each in duplicate, and the derived kinetic data were averaged before
calculation of group means.
Statistical analyses
Lineweaver-Burk plots were constructed for individual tissues
using least squares regression analysis, and the apparent
Km for corticosterone and the
Vmax derived from the line of best fit (20).
Differences among groups for apparent Km and the
Vmax were assessed by one-way ANOVA, and between
placental zones by paired t tests (21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Maternal adrenalectomy on day 13 had
no effect on placental weight on day 19 (either basal or labyrinth
zones). Following unilateral fetectomy on day 16, placentas were
retained in situ until collection on day 22 but weighed less
than those of untreated rats, whereas after fetectomy on day 19
placental weight on day 22 was similar to, but more variable than, that
of untreated rats (Table 1). Histological examination of placentas
revealed that fetectomy induced morphological changes in the labyrinth
zone, most notably increased edema, indicative of some degeneration,
and this effect precluded reliable dissection of the two zones.
Importantly, however, the size of the basal zone at day 22 appeared
unaffected by fetectomy 3 or 6 days earlier.
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; mRNA for the MR was not
detectable in placenta at any stage, although signal was clearly
evident in positive control kidney sections (Fig. 1j). Messenger RNA
for the GR was consistently expressed in the two placental zones and
remained effectively unchanged at the three stages of pregnancy
examined (Fig. 1, a and b); hybridization of placental sections with
the GR sense strand cRNA probe yielded effectively no signal (Fig. 1l).
In contrast to the GR, mRNA expression for both 11ß-HSD enzymes
varied markedly between days 16 and 22 in the two placental zones;
11ß-HSD-1 mRNA was evident in basal zone at day 16 then appeared to
fall by day 22, whereas its expression clearly increased over the same
period in labyrinth zone (Fig. 1, d and e); sense strand cRNA controls
yielded minimal signal, comparable with or less than that observed in
negative regions of placenta on the 2 days of pregnancy
(i.e. labyrinth zone at day 16 for 11ß-HSD-1 and day 22
for 11ß-HSD-2). Positive signal for 11ß-HSD-1 mRNA was observed
specifically in trophoblast cells within each placental zone (Fig. 2, a and b). The reverse pattern of
change occurred in the two placental zones with respect to mRNA for
11ß-HSD-2 (Fig. 1, g and h), consistent with changes in 11ß-HSD-2
bioactivity (see below). Messenger RNA for 11ß-HSD-2 was also
specifically localized to trophoblast cells, and this was especially
evident in the labyrinth zone at day 16 (Fig. 2c). Placental expression
of mRNAs for 11ß-HSD-1, -2, and GR on day 19 were all unaffected by
maternal adrenalectomy on day 13 (data not shown). Following fetectomy
(both Fx 16 and Fx 19), however, the presumptive labyrinth zone
appeared very low or negative for 11ß-HSD-1, -2, and GR mRNAs,
whereas expression of all three transcripts appeared relatively
unaffected in basal zone (Fig. 1, c, f, and i).
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). In contrast,
11ß-HSD-2 activity was maximal in labyrinth zone on day 16, remained
high on day 19, but then fell (P < 0.01) by day 22
(85% lower than day 16 value).
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).
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). The apparent
Km of the enzyme in fetectomized placentas was
not different from that in either basal or labyrinth zones of control
placentas, whereas the Vmax was intermediate
between that in basal and labyrinth zones. The
Vmax in whole placenta at day 22 was similar in
the Fx16 group (2.0 ± 0.4 pmol/min per mg protein) and the Fx19
group (1.7 ± 0.2), and neither differed significantly from the
value for whole placentas in control rats (2.2 ± 0.2 pmol/min per
mg protein; derived from separate estimates made for basal and
labyrinth zones and their relative weights).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distribution of mRNAs for the 11ß-HSD enzymes in the two placental zones identified by in situ hybridization is consistent with the pattern recently reported by Burton et al. (7) using S1 nuclease analysis. The striking feature of this pattern is that marked yet opposite changes occur in the expression of the two enzymes in the two placental zones over the final week of pregnancy. Thus, whereas mRNA for 11ß-HSD-2 virtually disappeared in trophoblast cells of labyrinth zone between days 16 and 22, it was at least maintained in those of the basal zone. Conversely, mRNA for 11ß-HSD-1 was effectively absent from the labyrinth zone on day 16 but then increased dramatically to day 22. These contrasting patterns of mRNA expression for 11ß-HSD-1 and -2 in the two zones are suggestive of distinct regulatory signals operating at these two locations. This may reflect differences in the supply of maternal blood to the two placental zones, which is directed initially to the labyrinth zone before passing to the basal zone (22). Indeed, because this vascular arrangement effectively represents a portal system, it raises the possibility that products of the labyrinth zone per se could influence gene expression in the basal zone, including that for the two 11ß-HSD enzymes.
The pattern of 11ß-HSD-2 bioactivity in the basal and labyrinth zones closely paralleled the mRNA expression for this enzyme. The observed apparent Km (in the order of 20 nM) is consistent with 11ß-HSD-2 bioactivity (23, 24) and was generally similar between the zones and with advancing pregnancy. One exception was the higher Km evident in basal compared with labyrinth zone at day 22, possibly reflecting higher levels of progesterone in the basal zone at this time. Progesterone is a potent inhibitor of 11ß-HSD (25, 26) and is synthesized locally within the basal zone of the rat placenta (18, 27), albeit at a relatively low level in vivo (28). In contrast to 11ß-HSD-2, bioactivity characteristic of 11ß-HSD-1 was not detectable in either placental zone at any stage of pregnancy, despite the presence of substrate concentrations up to and well beyond the known Km for this enzyme. This presumably reflects a loss of enzyme activity associated with cellular disruption during homogenization because Burton et al. (7) recently showed that 11-oxoreductase activity (generally ascribed to the 11ß-HSD-1 enzyme; see Refs. 29–31) in tissue fragments of rat placenta changed in parallel with 11ß-HSD-1 mRNA expression and immunoreactivity. An alternative explanation for the absence of 11ß-HSD-1-specific bioactivity is that the 11ß-HSD-1 mRNA detected by in situ hybridization in the present work is an alternative transcript that encodes a protein lacking enzyme activity as occurs in rat kidney (32). This seems unlikely, however, given the considerable 11-oxoreductase activity associated with 11ß-HSD-1 in placental fragments (7).
Colocalization of mRNA expression for the GR and the 11ß-HSD enzymes in basal and labyrinth zones suggests that placental 11ß-HSD could regulate glucocorticoid effects within the placenta. Although previous studies had identified glucocorticoid binding sites indicative of the GR in the placenta of several species including the rat (33, 34, 35), to our knowledge the present work provides the first evidence of mRNA expression for the GR in rat placenta. It was recently suggested by Karalis et al. (9) that the presence of 11ß-HSD in the human placenta may limit local bioactivity of maternal cortisol and thereby influence placental function(s). This is likely to be the effect of 11ß-HSD-2 in the rat placenta because this enzyme appears to act exclusively as an 11ß-dehydrogenase (23, 24) and as such would reduce levels of active glucocorticoid. On the other hand, the present study shows that 11ß-HSD-1 is also coexpressed with the GR in rat placenta, and so glucocorticoid bioactivity could even be enhanced at some stages of pregnancy, depending on the specific placental zone and the direction of the reaction catalyzed by 11ß-HSD-1. Although this form of the enzyme has the capacity for bidirectional activity, albeit inherently difficult to measure in homogenates as discussed above, 11-oxoreductase activity appears to be the dominant reaction in intact cells (29, 30, 31) and tissues (7, 36) including the basal and labyrinth zones of the rat placenta (7). The very different expression patterns for 11ß-HSD-1 and -2 in these adjacent placental zones also raises the possibility of a physiological interaction between the enzymes in relation to substrate supply, similar to that proposed for the uterine endometrium where the two enzymes are differentially expressed in epithelial and stromal cells (37).
With regard to specific glucocorticoid actions within the placenta, several important placental functions are affected by glucocorticoids including synthesis of peptide (9, 38) and steroid (39) hormones. There may also be a number of interactions between glucocorticoids and progesterone such as that recently demonstrated by Karalis et al. (9) in which the inhibition of CRH synthesis by progesterone in cultured human trophoblasts was blocked by cortisol. This effect of progesterone is thought to be mediated via progesterone interaction with the GR rather than the progesterone receptor (PR) because the latter is not detectable in human trophoblasts (9). Although the rat placenta does not produce CRH, progesterone does have other important effects on placental function in this species, most notably with respect to growth (40). Moreover, whereas the present study localizes mRNA for the GR to both placental zones, a previous report indicates that the PR is expressed only in basal zone (41). This raises the possibility that any progesterone effects in the labyrinth zone may be mediated via the GR, and as such would be susceptible to inhibition by glucocorticoids as occurs in human placental trophoblasts (9).
Two important species differences are evident with respect to the pattern of placental 11ß-HSD expression in the rat and the well characterized baboon model (for review, see Ref.10). Firstly, the pattern of change in the rat labyrinth zone (i.e. reduced 11ß-HSD-2 and increased 11ß-HSD-1 mRNA expression) is suggestive of a reduction in the placental glucocorticoid barrier between mother and fetus, whereas this barrier is clearly enhanced late in baboon pregnancy (42, 43). Secondly, fetectomy studies in the baboon show that unlike observations of the present work, the shift in placental 11ß-HSD activity with advancing pregnancy is dependent on the presence of the fetus (44), specifically through its supply of adrenal androgens for placental estrogen synthesis (26). Indeed, this enhancement of the placental glucocorticoid barrier near term appears to activate maturation of the baboon fetal HPA axis and thus promote fetal autonomy (10, 43). The present study in the rat, however, shows that premature activation of the fetal HPA axis had no effect on 11ß-HSD-2 bioactivity in either basal or labyrinth zones, and fetectomy had no apparent effect on mRNA expression for either 11ß-HSD-1 or -2 in the basal zone. Moreover, 11ß-HSD-2 bioactivity in whole placenta on day 22 of pregnancy was unaffected by fetectomy 3 or 6 days earlier. Fetectomy did prevent the marked increase in 11ß-HSD-1 mRNA expression in labyrinth zone, but the biological significance of this effect remains uncertain because there was considerable degenerative change in this zone post fetectomy, consistent with previous analyses of placental morphology after fetectomy (45). It is noteworthy in this regard that GR mRNA expression was also absent throughout most of the labyrinth zone after fetectomy, presumably reflecting loss of trophoblast, fetal mesenchyme, and fetal vascular tissues. One reason why the influence of the fetus on placental 11ß-HSD may be so different in the rat and baboon is the presence in the latter of a feto-placental unit for estrogen synthesis. Thus, the primate placenta is dependent on a continuous and increasing supply of fetal adrenal androgens to ensure rising estrogen levels near term (for review see Ref.10), and because the trophic drive for this fetal androgen production is derived partly from fetal pituitary ACTH, loss of the placental glucocorticoid barrier could block this trophic support and thereby compromise placental estrogen synthesis. In contrast, the rise in maternal estrogen observed near term in the rat is ovarian in origin (46) and thus not dependent on fetal adrenal status. One could postulate, therefore, that evolution of the feto-placental unit for estrogen synthesis, which is unique to higher primates, required an associated change in the pattern of placental 11ß-HSD expression.
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Acknowledgments |
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Footnotes |
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Received August 4, 1997.
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