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Department of Biological Chemistry (J.A., J.O.), The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; and the Division of Reproductive Biology (A.H.P.), Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305
Address all correspondence and requests for reprints to: Dr. Joseph Orly, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. E-mail: orly{at}vms.huji.ac.il
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Introduction |
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The biosynthesis of steroid hormones has been studied extensively in "classical" steroidogenic cells of the adrenal cortex (15), and steroidogenic cell types of the gonads (16, 17). Steroidogenesis in the human placenta has been studied extensively, because of the ready availability of this tissue for research (18, 19, 20, 21). By contrast, the endocrine capacities of the pregnant uterus in rodents are less well understood (22), and probably have been overlooked, because during pregnancy in rodents, progesterone and estrogen are produced mainly by the ovary (23). Therefore, surgical removal of the ovary at any stage during mouse and rat pregnancy results in termination of the pregnancy (18). In contrast to the human placenta, during the second half of pregnancy, production of estrogen in the rodent ovary depends on androgens that are synthesized in the placenta starting on days 10–11 post coitum (24). Thus, the newly formed rodent placenta, which expresses P4501c17 is the major site for the conversion of the ovarian-synthesized progestins into androgens, which then serve as the substrate for aromatization in the ovary.
Probably biased by our understanding of placental endocrinology in humans, previous attempts to characterize steroidogenic capacities in the pregnant uterus of rodents focused on the second half of gestation, when the placenta is fully developed (24, 25, 26, 27). Most of the studies employed the rat as a model system. For example, progesterone production by minced placental preparation was low on day 10, peaked on day 12, and declined to low levels until term (25). In accordance with these results, the expression of P450scc, was shown to dramatically increase between days 10 and 12, and declined thereafter (24). A similar pattern was observed for 3ßHSD, expression of which is essential for the production of progesterone and further hormone products. Biochemical and histochemical analyses, conducted in the early 1960s, suggested that the activity of 3ßHSD in rat placenta was present exclusively in the giant trophoblast cells (22, 28, 29, 30).
The introduction of more sensitive methods, such as RT-PCR, as well as in situ hybridization to study the ontogeny of P450scc expression in pregnant uterine tissue, has recently revealed novel insights that could potentially modify earlier notions regarding the role of local steroidogenic capacities during pregnancy in rodents. First, we have shown that implantation of the blastocyst initiates expression of P450scc messenger RNA (mRNA) in the decidualizing stroma (31). Moreover, in situ documentation of P450scc mRNA suggested that the decidua expression of this gene correlates with the fate of the decidua tissue, which gradually degenerates in mice as of day 8.5–9.5 post coitum. At this time, a rise in P450scc was observed in the giant trophoblast cells, which express high amounts of P450scc mRNA between days 8.5–10.5 (31). Other than the observation of decidual P450scc, the second novel finding that highlights the uniqueness of the uterine steroidogenic machinery is the identification of a 3ßHSD uterine-specific isoform, designated murine type VI (32). RT-PCR characterizations suggested that the 3ßHSD VI transcript is expressed in cells of the implantation site during the first half of mouse pregnancy (32).
The aim of the present study was to establish that the decidua cell layers acquire all the necessary components essential for the production of progesterone by expressing P450scc and its accessory proteins, StAR and 3ßHSD VI. To accomplish this we compared the levels of the steroidogenic gene products to those expressed in the giant trophoblast cells. Our data suggest that the steroidogenic potential to locally produce steroid hormones in the pregnant uterus is first initiated in the differentiating decidua cells and culminates on days 8.5–9.5 p.c., when a few layers of giant trophoblast cells express high amounts of the steroidogenic components essential for the production of progesterone. However, as might be concluded from the profiles of P450scc, StAR and 3ßHSD VI, the capacity for de novo production of steroid hormones in the mouse uterus is abruptly lost beyond day 11, indicating that mature placental tissue during the second half of mouse pregnancy does not contribute significantly to steroidogenesis.
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Materials and Methods |
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-32P]Deoxy-dCTP (no. PB10205),
35S-methionine (no. SJ235) were purchased from
Amersham Pharmacia Biotech (Little Chalfont, UK). T4 DNA
Ligase no. 481220, collagenase/dispase (Vibrio alginolyticus/Bacillus
polymyxa) was from Roche Molecular Biochemicals, Mannheim,
Germany. DMEM and Ham’s F-12 were from Grand Island Biological (Grand
Island, NY).
Immunoreagents
Rabbit P450scc antiserum to rat P450scc was prepared and
previously characterized (33). Rabbit antiserum raised against the
human placental 3ßHSD was kindly provided by Dr J. Ian Mason. Other
antisera included commercially available lissamine rhodamine-conjugated
AffiniPure goat antirabbit IgG (H + L) and peroxidase-conjugated
AffiniPure goat antirabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Protein A-Sepharose (no.
9424), 3,3'-diaminobenzidine (DAB, D-4168) and ExtrAvidin-peroxidase
(EXTRA-1) were from Sigma (St. Louis, MO). ELF kit
(E-6605) was purchasd from Molecular Probes, Inc. (Eugene,
OR).
Animals
Intact, immature female Sprague Dawley rats (21 days old) and F1
hybrids of C57XBalb/C mice were obtained from Harlan
(Jerusalem, Israel) and maintained under 16-h light, 8-h dark schedule
with food and water ad libitum. Animals were treated in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals. All protocols had the approval of the Institutional Committee
on Animal Care and Use, The Alexander Silverman Institute of Life
Sciences, The Hebrew University of Jerusalem. To obtain high fecundity
rate, 6- to 8-week-old C57xBalb/C F1 females were hyperstimulated by ip
injection of 5 IU PMSG at noon, and 4 IU hCG was administered 48 h
later. Mating was allowed during the following night by placing one
sexually trained Balb/C male per two females. In the
morning, animals were examined for vaginal plugs and noon time was
determined as day 0.5 p.c., or E0.5. At the indicated times, mice
were killed by cervical dislocation, and the uterus horns were removed
and placed in PBS. In preparation for primary cell cultures, the tissue
was placed in DMEM:F-12 culture medium.
Uterine tissue preparation
Uterine tissue was trimmed free of fat and processed for further
use in the following manner: whole uterine horns were used for in
situ hybridization and immunohistochemical analyses; whole uteri
from 3.5- and 4.5-day pregnant animals were used for RNA and protein
preparations after validating pregnancy by flushing the horns with PBS
to recover blastocysts; E6.5 and E7.5 implantation sites were
mechanically separated from the myometrium, and the fetuses were
surgically removed; the embryos were also removed from E8.5- E10.5
implantation sites before further processing for RT-PCR or Western blot
analyses. When indicated, the giant trophoblast cells and the placental
cell layers were gently removed by scraping the inner face of
hemi-implantation sites (E9.5) with a small round-tip spatula. The
remaining myometrium-free cell types, designated decidua, constituted
the decidua capsularis, decidua basalis and a minor contamination of
stromal cells.
Cloning of mouse P450scc complementary DNA (cDNA) by 3'
RACE-PCR
A 3' RACE protocol was performed as described (34) including the
following modifications: total RNA was reverse transcribed using 5 pmol
of a primer adapter 5'-GGCCTCTAGACAGCATTTATGTGTTTATTAA-17dT-3'. The
resulting cDNA was amplified using a primer sequence corresponding to a
previously published (35) 5' mouse untranslated region
(5'-GGCCAAGCTTAGAGACACTTGTGCAGCA) and the above mentioned primer
adapter lacking the poly dT. The primers included convenient
HindIII and XbaI sites at their 5' end,
respectively. RT reaction was performed using 1 µg of mouse E6.5
decidual RNA and the resulting cDNA was ethanol precipitated before PCR
amplification. PCR was conducted for 40 cycles using VentR
DNA polymerase (New England Biolabs, Beverly, MA) and the
manufacturer supplied buffer including 4 mM of
MgCl2. The PCR product was purified from agarose gel by use
of GeneClean II (Bio 101 Inc., La Jolla, CA) and digested with
restriction enzymes before ligation into KS Bluescript vector
(Stratagene, La Jolla, CA). Three clones recovered from
three independent RACE-PCR reactions were sequenced by primer walking
using automated fluorescent cycle sequencing with dye labeled
terminators and PE Applied Biosystems instrumentation
(ABI377 PE Applied Biosystems, Foster City, CA).
RT-PCR analysis
Relative-quantitative RT-PCR procedure was performed as
previously described (36). In brief, total RNA was prepared by RNAzol B
extraction according to the manufacturer’s instructions. Aliquots
containing 200 ng RNA were assayed by RT followed by PCR. The PCR was
conducted in the presence of 2 µCi of
[-32P]-deoxy-ATP (3000 Ci/mmol), all four dNTPs (200
mM) and 500 ng of the appropriate oligonucleotide primers
(50–60 pmol); oligonucleotide primers for the ribosomal protein L19
served as an internal control. Following the PCR reaction (20 cycles,
annealing temperature of 65 C), tracking dye gel buffer was added to
20–40 µl of the PCR reaction mixture for further analysis by PAGE
(36). The dried gels were quantified using a Fuji Photo Film Co., Ltd. Bio-Imaging analyzer (BAS-1000, Fuji Photo Film Co., Ltd. Film, Tokyo 106, Japan). The radioactivity in each of the
PCR bands was normalized to the radioactivity of the ribosomal protein
L19 band. Gels were also exposed to RX medical x-ray film (Fuji Photo Film Co., Ltd. Film, Tokyo 106, Japan) for 2–16 h at -80
C and developed by an X-Omat processor.
PCR oligonucleotide
PCR oligonucleotide primer pairs (A, forward; B, reverse) were
designed based on known cDNA sequences of the various target genes.
Mouse P450scc:
A, 5'-AGAAGCTGGGCACTTTGGAGTCAG
B, 5'-TCACATCCCAGGCAGCTGCATGGT
Mouse P450scc for full length cDNA (1–1792):
A, 5'-GGCCAAGCTTAGAGACACTTGTGCAGCA
B, 5'-GGCCTCTAGCCAGTGTTCAAGTGTTTATTAA
Mouse 3ßHSD I and VI (12):
A, 5'-ACTGGCAAATTCTCCATAGCC
B, 5'-CCACTTGTCAACTGGGAGGAA
Rat P450c17 (37):
A, 5'-GGGGCAGGCATAGAGACAACT
B, 5'-GCCTGAGCGCTTCTTAGATCC
Rat StAR (3):
A, 5'-GCAGCAGGCAACCTGGTG
B, 5'-TGATTGTCTTCGGCAGCC
Rat ribosomal protein L19 (38):
A, 5'-CTGAAGGTCAAAGGGAATGTG
B, 5'-GGACAGAGTCTTGATGATCTC
Mouse ferredoxin (39):
A, 5'-GACTCTCTGCTAGATGTTGTG
B, 5'-GCTCATGTCAACAGACTGTCG
Mouse NADPH-ferredoxin reductase (39):
A, 5'-GCTGTGGAGTATGAAGCGAAG
B, 5'-ATAGTCCACAGCCTGGGTGTG
Primary cell cultures
Decidua cells. Decidual and stromal cells were isolated from
uterine tissue on day 6.5 of pregnancy. Uteri were removed and several
implantation sites were mechanically separated from the myometrium.
After removing the embryo, the remaining tissue was cut into small
pieces and incubated in culture medium (serum free) containing 10 mg/ml
BSA, 4 mg/ml collagenase/dispase and 100 µg/ml DNase I (500 mg tissue
in 5 ml). Following 15 min incubation at 37 C, the cells were dispersed
by gently pipetting the minced tissue fragments through a P1000 Gilson
tip, or a glass Pasteur pipette. This process was repeated three times,
the tissue remains were discarded and two volumes of medium containing
10% FCS were added. The cell pellet was collected following 2 min
centrifugation at 200 x g (5 min). The cells were
seeded into a 24-multiwell plate (Nunc Copenhagen, Denmark) and grown
in 0.5 ml DMEM supplemented with 10% FCS. Incubations were at 37 C in
a humidified incubator, 95% air, 5% CO2.
Giant trophoblast cultures. The layers of the giant trophoblast cells including the placental zones were removed with a spatula from E9.5 hemi-implantation sites as described above. Further dispersion to single cell suspension was performed by use of the collagenase-dispase cocktail, as described above. Cultures were maintained in DMEM:F-12 medium (0.5 ml) containing 10% FCS.
Granulosa cell cultures. Granulosa cells were prepared and grown in serum-free medium in 24-multiwell plates as previously described (40). Twenty-four hours before pulsing with 35S-methionine (described below), FSH (100 ng/ml) was added to induce P450scc expression.
Western blot analysis and imunoprecipitation of metabolically
labeled proteins
Western blot analysis was performed exactly as previously
described (41). Metabolic labeling was performed according to Hales
et al. (42). Briefly, methionine starvation was performed
16 h after seeding by a 2 h incubation with methionine-free
DMEM, followed by a 2 h pulsing with 1.5 µCi of
35S-methionine added in 250 µl of the same medium. The
cells were then washed twice with PBS and harvested in RIPA buffer
containing 6 mg/ml methionine. After 30 min incubation on ice the
samples were centrifuged for 1 min at 14,000 x g and
the supernatant was collected for further immunoprecipitation in a
final volume of 500 µl RIPA. Anti-P450scc serum (1:1000) was added
for 2 h incubation at 4 C. The immunocomplex was harvested by
adding 20 µl of protein A-Sepharose (1:5 vol/vol suspension in PBS)
for 1 h incubation at 4 C using a revolving carousel. The
Sepharose beads were pelleted by a short spin (200 x
g), followed by a brief wash with 1 ml PBS containing 0.1%
Tween-20 (PBST). Finally, 30 µl of SDS-PAGE sample buffer containing
ß-mercapto-ethanol were added and the samples were boiled for 2 min.
Samples were electrophoresed on 10% SDS-PAGE, dried and exposed to
RX medical x-ray film (Fuji Photo Film Co., Ltd. Film, Tokyo 106, Japan) for 16 h at -80 C.
Measurments of 3ßHSD activity
Enzyme activity was determined by measuring the conversion of
[7-3H]-pregnenolone (0.5 µM; 3 µCi/ml,
NET-039 22.6 Ci/mmol, NEN Life Science Products, Boston,
MA) to 3H-progesterone by the cultured cells (43). Briefly,
decidual cells (
3 x 104), or giant trophoblast
cells (1 x 103) were seeded into 16-mm wells of a
24-multiwell plate, as described above. On the morning after seeding,
radioactive pregnenolone was added to the culture medium (0.5 ml) in
the absence or presence of cyanoketone (5 µg/ml). One hour later the
cells were lysed by adding ethanol (final concentration 20%) and the
total content of the steroids was extracted by 5 vol ether (43). TLC of
the extracted 3H-steroid products was performed (43) using
none-labeled authentic pregnenolone and progesterone as internal
standard markers (43). Each experimental lane was cut into 50 fractions
and the relative radioactivity (% of total cpm) was presented. The TLC
patterns (see Fig. 7
) represent two independent experiments with
similar results.
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Immunocytochemical staining
The immunofluorescence staining procedure used to visualize
mitochondrial P450scc in primary cultures of decidua or giant
trophoblast cells has been previously described for granulosa cell
cultures (44). Briefly, culture monolayers were fixed in
paraformaldehyde PBS solution, Triton X-100 permeabilized, and
immunofluorescently stained by use of polyclonal rabbit antiserum rat
P450scc (1:50) and lissamine-rhodamine-labeled goat (IgG) antirabbit
IgG (1:20).
Electron microscopy
Intact uteri, trimmed free of fat and small fragments (1
mm3), were fixed in freshly prepared PBS containing 1%
glutaraldehyde and 3% p-formaldehyde (33). After overnight incubation
at 4 C, preparation of the tissue blocks in LR White resin (London
Resin Co., Bassingstoke, UK) was performed as recently described (7).
Thin sections were incubated with 1:20 dilution of rabbit antirat
P450scc, followed by incubation with 1:10 dilution of gold-labeled goat
antirabbit IgG (7).
In situ hybridization
In situ hybridization was performed as described by Lev et
al. (45). Briefly, uteri were removed and fixed for 12 h at
room temperature in freshly prepared 4% p-formaldehyde solution. After
embedding in paraffin, 7 µm sections were cut and mounted on
microscope slides (SuperFrost/Plus, Menzel Glaser, Germany). After air
drying for one hour at 37 C, slides were stored at -20 C until further
use. For detection of 3ßHSD type VI, or I, we used a 5' biotinylated,
2'-O-methyl-RNA 5'-AUGACACCUG UGACAUCAAU GACAGCAGCA
GUGUGGAUGA CAACAGAGAU-3' probe (custom made by Microsynth,
Switzerland).
Slides were deparaffinized by heating to 60 C for 1 h, followed by 2 changes of xylenes (10 min each), two washes in ethanol and rehydration by consecutive 2 min immersions in ethanol solutions in PBST (100%, 75%, 50%, 25%, and 0%). Thereafter, slides were treated at room temperature with 10 µg/ml proteinase K in PBST for 15 min, followed by one wash in 2 mg/ml glycine PBST and twice with PBST (5 min). Prehybridization was performed by adding prehybridization buffer (200 µl/slide) containing 50% formamide, 5 x SCC, pH 4.5, 50 µg/ml yeast transfer RNA and 50 µg/ml heparin. Slides were incubated for 1 h at 60 C. Hybridization was performed by overnight incubation at 60 C in prehybridization buffer containing 10 µg/ml probe. Posthybridization washes (30 min) were performed as follows: first, two room temperature washes in solution 1 (50% formamide 5 x SSC, pH 4.5, and 0.5% SDS), and then two washes in solution 2 (50% formamide 2 x SSC, pH 4.5) performed at 60 C (30 min). Finally, slides were washed 3 times (room temperature) in TBST containing 250 mM Tris-HCl, pH 7.5, 1.3 M NaCl, 30 mM KCl, 1% Tween-20 and 2 mM levamisole (Sigma L-9756). The last wash was performed for 60 min in blocking solution (1% skim milk in TBST). The bound probe was detected by strepavidin-alkaline phosphatase conjugate using ELF kit according to the manufacturer’s instructions.
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Results |
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shows the sequence of the cloned mouse P450scc cDNA and its deduced
amino acids. The full-length sequence of this cDNA measures 1791
nucleotides, a short 5' UTR of 42 nucleotides, an open reading frame of
1578 nucleotides encoding 526 amino acids, and a 168-nucleotide long 3'
UTR. The mouse and the rat cDNA sequences show 91% homology, whereas
their deduced amino acid sequences show 88% homology.
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). These results
confirmed our previous in situ hybridization and Northern
blot analyses showing that expression of P450scc transcript in the
decidua was induced upon implantation and peaked at day 9.5 post
coitum. To a large extent, the expression pattern of the
uterus-specific isoform of 3ßHSD VI paralleled the temporal changes
of the P450scc transcript. A very low level of 3ßHSD VI mRNA was
detectable in the pregnant uterus before implantation (Fig. 2C), with a
12-fold rise on day 4.5, the day of implantation, and reaching a peak
on day 9.5 of pregnancy. Unlike the gradual increase of the P450scc
mRNA content per implantation site, the marked stepwise increase of
3ßHSD VI transcript on day 8.5 correlated well with the appearance of
the giant trophoblast cells, as supported by the concomitant expression
of P450c17 (Fig. 2D). Unlike P450scc and 3ßHSD VI, which were
expressed in the decidual cells of E9.5 sites (Fig. 2B), P450c17 is
evidently a typical marker of the embryonal trophoblast cells, as
suggested from microsurgical purification of these cells from E9.5
implantation sites (Fig. 2E). These results implied that the ascending
levels of P450scc and 3ßHSD VI mRNAs detected during days 4.5–8.5
were probably expressed by the decidua tissue before the appearance of
the secondary giant trophoblast cells.
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). Compared with the level of P450scc
in corpora lutea, the cytochrome levels in the uterus were markedly
low. Unlike the readily observed induction of P450scc mRNA upon
implantation on day 4.5, the protein could not be detected before day
6.5 (Fig. 3A). Thereafter, faint protein signals obtained during days
6.5–7.5 suggested the apparent presence of P450scc in the decidua
tissue. The decidua capacity to express P450scc protein could be
further substantiated by a metabolic labeling study of decidual cell
dispersates, showing immunoprecipitable 35S-methionine
labeled P450scc in this cell type (Fig. 3B). Again, metabolic labeling
of P450scc in control cultures of prepubertal granulosa cells treated
with FSH clearly demonstrated that the decidua expression of P450scc
was relatively limited. As pregnancy progressed, the protein levels of
P450scc peaked between days 8.5–9.5 (Fig. 3A), in accordance with a
similar pattern observed for the mRNA profile.
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, C–D). No
immunostaining for P450scc protein was detected in E9.5 decidua
capsularis or decidua basalis (Fig. 3D).
Using different approaches, including immunocytochemical and
immuno-electron microscopy, a relatively weak immunofluorescent
staining of mitochondria was demonstrated in cultured decidua cells
from day 6.5 implantation sites (Fig. 4A). The significance of the low P450scc
staining patterns in the decidual cell mitochondria could be assessed
by comparison to naive ovarian granulosa cells examined before and
after FSH treatment. Clearly, untreated control monolayers were devoid
of any mitochondrial signals (Fig. 4F), whereas bright mitochondrial
P450scc immunofluorescent signals were typical for FSH-treated cells
(Fig. 4G). Due to the relative short exposure to FSH (16 h), some of
the granulosa cells were less responsive and expressed less P450scc
(Fig. 4G), similarly to the immunoreactive enzyme levels observed in
the decidual cells (Fig. 4A).
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). The low gold labeling of the decidual
mitochondria is consistent with the lamellar formation of the cristae
(Fig. 4B), typical of an early-differentiating steroidogenic cell (33).
By contrast, the highly steroidogenic mitochondria of the E9.5 giant
trophoblast cells (Fig. 4, C and D) were heavily decorated by
immuno-gold particles in the vesicular structures of the mitochondrial
cristae (Fig. 4D). Thus, the ultrastructure of the giant cell
mitochondria is similar to characteristic terminally differentiated
mitochondria in cells of the adrenal cortex,
interstitial cells of the ovary and corpus luteum (33).
Uterine 3ßHSD VI
In light of the progressive accumulation of 3ßHSD VI mRNA during
early pregnancy (Fig. 2), we examined the expression of this transcript
and its encoded protein at the cell-specific level. We first performed
in situ hybridization and immunohistochemical
characterization of 3ßHSD VI in sections of E6.5 and E7.5
implantation sites. Figure 5, B–D, shows
that 3ßHSD VI mRNA and protein are exclusively expressed in the
decidua tissue and excluded from the nondifferentiated stroma, or the
embryo. Moreover, on days 6.5–7.5 of embryonal development, a few
secondary giant trophoblast cells can be seen upon their migration from
the ectoplacental cone toward the antimesometrial pole of the
implantation site, as can also be seen in Fig. 5, D–E. Clearly,
3ßHSD VI protein was not expressed in these migrating cells observed
on day 7.5 (Fig. 5E). However, by E9.5, the giant trophoblast cells
exhibited an enormously high accumulation of the enzyme (Fig. 6B). The exclusive expression of 3ßHSD
VI in the giant cells was contrasted by its lack in the embryo, the
decidual zones, the stroma and even cells of the primitive placenta and
the allantois (Fig. 6, B–D).
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). This activity is readily inhibitable
by cyanoketone, an inhibitor of 3ßHSD. Giant trophoblast cell
cultures displayed a higher activity of 3ßHSD, which corroborates the
strong immunoresponsive fluorescence signals obtained for this protein
in the extraembryonic cell type.
Western blot analysis (Fig. 8) confirmed
that the giant trophoblast cells indeed express the VI isoform of
3ßHSD, which migrates more slowly than the ovarian type I isoform of
the enzyme (32). Evidence is provided in Figs. 6, F–G, and 8, that
3ßHSD VI is not expressed in the developing placenta at
mid-pregnancy. Western analyses showed that the total level of 3ßHSD
VI dropped precipitously at day 10.5 (Fig. 8) and the enzyme was no
longer expressed in the mature placenta on day 14.5 (Fig. 6, F–G). The
latter immunohistochemical observations showed that only the few
degenerating giant cells still exhibited some expression of 3ßHSD VI,
whereas the rest of the placental cell types, including the
spongiotrophoblasts and the labyrinth cells, were devoid of this key
steroidogenic enzyme. Moreover, the abrupt loss of 3ßHSD VI during
development of the mature placenta was accompanied by a concomitant
loss of P450scc and StAR proteins, which were markedly decreased
commencing on day 10.5 of pregnancy (Fig. 8).
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, lane 2). It is also apparent that as
pregnancy progressed, the expression of the accessory proteins declined
concomitantly with the loss of P450scc. Thus, StAR and ferredoxin
reductase mRNA levels were nearly null in decidua of day 9.5 (lanes 5).
Surprisingly, the ferredoxin signal was fairly high in the E9.5 decidua
cells. However, our results suggest that ferredoxin is probably not an
exclusive marker of steroidogenic capacity because it was also
expressed in the E9.5 embryo and the preimplantation uterus of day
3.5 p.c., which does not express P450scc, StAR, and ferredoxin
reductase. Finally, as expected, the highest levels of StAR, ferredoxin
and ferredoxin reductase transcripts were expressed in the E9.5 giant
trophoblast cells, consistent with their P450scc content (lanes 4) and
their well established steroidogenic capacity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study focused on the spatio-temporal expression of 3ßHSD VI during early mouse gestation. At selected time points, we also demonstrated the expression of P450scc and its accessory proteins, which are required for de novo synthesis of steroid hormones. The latter include mitochondrial ferredoxin and ferredoxin reductase, which facilitate electron flow from NADPH+ to P450scc, and StAR, a protein that facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where P450scc is found (3, 4, 48, 49, 50). Collectively, our data suggest that the expression patterns of these genes are precisely coordinated, in both time and location. For example, whereas implantation initiates the expression of P450scc and 3ßHSD VI in decidua of days 4.5- 7.5 p.c., the level of both gene products concomitantly declines in this tissue during days 8.5–9.5 of gestation, so that no 3ßHSD VI protein was observed on 9.5 day p.c., and only a relatively low level of P450scc mRNA was seen on 9.5 day in decidua tissue (31). At this time in mouse pregnancy, expression of the steroidogenic enzymes shifts to the giant trophoblast cell layers, which also become endowed with StAR and ferredoxin/ferredoxin reductase. Moreover, the giant trophoblast cells are the only cell type containing numerous lipid droplets and round mitochondria with typical vesicular cristae known as the hallmark cytological markers of a classical steroidogenic cell (33). Interestingly, in contrast to the giant trophoblast cells, the underlying cells of the early placenta and the allantois are devoid of the steroidogenic gene products (this study and Ref. 31). As pregnancy progresses, Western analyses and immunohistochemical evidence suggest that the true placenta which develops to maturity during days 10.5–14.5 of gestation does not develop the capacity for de novo steroidogenesis. Thus the observed decline of P450scc, 3ßHSD VI, and StAR occurs concomitantly with the degeneration of the giant cell layers.
A combination of sensitive in situ hybridization and histochemical studies using the ELF-immunofluorescence staining method allowed for the demonstration of 3ßHSD mRNA and protein during the first half of mouse pregnancy. Experiments measuring the conversion of 3H-pregnenolone to 3H-progesterone demonstrated 3ßHSD activity in decidual cell cultures. Detection of P450scc protein in the decidua was more difficult but was accomplished through the use of immunoprecipitation. The relatively low content of the protein components necessary for the steroidogenic machinery in the decidua cells of the mouse uterus is not surprising considering that even in the human placenta, which at term produces 300 mg progesterone per day (18), the cellular level of P450scc is no more than 1/20 of that found in mid-term corpus luteum (51). What, then, is the physiologic role for local production of progesterone at the implantation site in the face of high serum levels of progesterone originating from the corpus luteum? Despite this seemingly paradoxical scenario, the apparent lack of any known mutation of the human placental isoform of 3ßHSD I (52, 53) suggests that progesterone produced early during the first trimester in trophoblast cells acts in a paracrine manner on the nearby decidua cells to prevent rejection of the fetus. There are a number of reports suggesting an immunologic role for progesterone at the implantation site. An immunosuppresant role for progesterone at the fetal maternal interface was suggested many years ago by Siiteri and colleagues (54, 55). More recent studies demonstrated the critical role of progesterone as an antiinflammatory agent in the mouse uterus (56, 57). A highly relevant study in human patients, implicating the need of production of progesterone by trophoblasts for maintenance of pregnancy during the first trimester of gestation was reported recently (58). Piccinni et al. described a decreased production of leukemia inhibitory factor (LIF), IL-4 and IL-10 by decidual T cells from women with unexplained recurrent abortions (URA) during the first trimester in comparison with the production of these cytokines in women with normal gestations. The URA in the women could not be explained on the basis of conventional criteria, including serum concentration of gonadotropins and steroids. The addition of progesterone to cultures of decidual T-cells from the URA women resulted in a marked increase in the development of LIF and IL-4-producing cells. The reduced production of IL-4 and IL-10 by decidual T cells in women with URA was not seen in their peripheral blood indicating that this is not an inherent feature of their T cells, but an alteration affected by the microenvironment in the uterus, i.e. lack of uterine progesterone (58). These data support our hypothesis that local production of progesterone, as suggested by this study, plays an important if not critical role in the development and maintenance of early pregnancy.
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Acknowledgments |
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Footnotes |
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Received April 26, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-4-isomerase from
placenta: expression in nonsteroidogenic cells of a protein that
catalyzes the dehydrogenation/isomerization of C21 and C19 steroids.
Endocrinology 126:2493–2498 5- 4-isomerase in
human adrenals and gonads. Mol Endocrinol 5:1147–11575-4 isomerase (3ß-HSD) family. The exclusive
3ß-HSD gene expression in the skin. J Biol Chem 268:19659–196685-4
isomerase and differential expression in gonads, adrenal glands, liver,
and kidneys of both sexes. Proc Natl Acad Sci USA 88:8870–88745 4 isomerase in human
placenta and fetal membranes throughout gestation. J Clin
Endocrinol Metab 75:956–961[Abstract]
and P450SCC gene expression and
regulation in the rat placenta. Endocrinology 130:1309–1317-hydroxylase/C17–20
lyase, and P450 aromatase in fetal mouse gonads. Endocrinology 135:262–268[Abstract]
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