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Endocrinology Vol. 140, No. 11 5220-5232
Copyright © 1999 by The Endocrine Society

ARTICLES

Expression of Steroidogenic Genes in Maternal and Extraembryonic Cells During Early Pregnancy in Mice1

Jonathan Arensburg, Anita H. Payne and Joseph Orly

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ontogeny and functional role of steroidogenesis during early gestation in rodents is poorly understood. In previous studies, we have shown that expression of messenger RNAs (mRNAs) encoding two key enzymes indispensable for de novo synthesis of steroid hormones, i.e. cholesterol side chain cleavage cytochrome P450 (P450scc) and a newly identified isoform of murine 3ß-hydroxysteroid dehydrogenase/isomerase type VI (3ßHSD VI), is initiated upon decidualization of the uterine wall induced by implantation. In situ hybridization and immunohistochemical visualization of 3ßHSD VI mRNA and protein shows high expression of this enzyme in the antimesometrial cells of the decidua of days 6.5–7.5 post coitum (p.c.). Thereafter, expression of 3ßHSD VI in the decidual zones disappears and is replaced by a high expression of mRNA and protein in the embryonal giant trophoblast cells. At the peak of their development on day 9.5 p.c., the mouse giant trophoblast cells also express Steroidogenic Acute Regulatory (StAR) protein, which is required for steroidogenesis in the gonads and adrenal cortex. Our findings also suggest that the declining levels of P450scc, 3ßHSD VI, and StAR proteins between days 10.5–14.5 p.c. in the developing placenta is consistent with previous reports that the mouse placenta is not involved in de novo synthesis of steroids during the second half of pregnancy. Collectively, the results of the present study suggest that, during early phases of pregnancy, local progesterone synthesis in the maternal decidua and the trophoblast layers surrounding the embryonal cavity is important for successful implantation and/or maintenance of pregnancy. We propose that the local production of progesterone acts as an immunosuppressant at the fetal maternal interface preventing the rejection of the fetal allograft.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
D E NOVO synthesis of steroid hormones requires several essential components: in the mitochondria, cholesterol side chain cleavage cytochrome P450 (P450scc), the first key steroidogenic enzyme, converts cholesterol to pregnenolone (1, 2). This reaction is catalyzed in the presence of atmospheric oxygen and reducing equivalents provided by an electron chain consisting of ferredoxin, ferredoxin reductase, and NADPH. Transfer of cholesterol substrate into the inner mitochondrial membranes to the site of P450scc is facilitated by the recently discovered protein, Steroidogenic Acute Regulatory (StAR) protein (3, 4, 5). Pregnenolone passes out of the mitochondria and is further metabolized to progesterone by 3ß-hydroxysteroid dehydrogenase/isomerase (3ßHSD) (1, 2). Noteworthy, although 3ßHSD is considered as an endoplasmic reticulum enzyme, recent studies have demonstrated that, at least in the steroidogenic cells of the adrenal, a substantial fraction of 3ßHSD colocalizes with P450scc in the inner-membrane of the mitochondria (6, 7). Each of the proteins involved in the production of pregnenolone, i.e. P450scc, StAR, ferredoxin, and ferredoxin reductase, is a product of a single gene. In contrast, 3ßHSD exists as multiple isoforms in man (8, 9, 10), rats (11), mice (12), and hamster (13). In the mouse, six tissue-specific isoforms, each a product of a distinct gene, have been identified (14).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
DNase I (D-0251), HEPES (P-3375), hydrocortisone (H-4001), insulin (I-5500), phenylmethylsulfonylfluoride (PMSF), poly-L-lysine (P-1399), proteinase K (P-0390), pronase (P-5147), RNase A (R-4875), transferrin (T-4515), yeast transfer RNA (R-9001), were obtained from Sigma (St. Louis, MO). PMSG and hCG are products of Gestil (Organon, Oss, Holland). [{alpha}-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 [{alpha}-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. 7Go) represent two independent experiments with similar results.



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  		Figure 7. Conversion of pregnenolone to progesterone by decidua and giant trophoblast cell 3ßHSD. Decidua and giant trophoblast cell cultures were prepared on days 6.5 and 9.5 p.c., respectively (in Materials and Methods). On the day after seeding, 3H-pregnenolone (0.5 µM) was added in the absence or presence of cyanoketone (5 µg/ml). After 1 h incubation, the cells and culture medium were extracted and analyzed by TLC as described in Materials and Methods. Authentic nonradioactive steroids (Preg., pregnenolone; P, progesterone) marked the fraction numbers containing the radioactive tracers.

 
Enzyme-linked imunohistochemical procedure
Uterine horns were removed, trimmed free of fat, and fixed overnight at 4 C in 4% p-formaldehyde in PBS. Paraffin embedding and sectioning (6 µm) were performed according to standard procedures. Sections were deparaffinized by heating to 58 C for 15 min, followed by an immediate immersion into xylenes and two changes of the organic solvent (5 min each). Rehydration was performed by washing in ethanol (x2) and consecutive 2 min immersions in 100%, 90%, and 80% ethanol, followed by a 30-sec rinse in running water. Finally, slides were transferred to PBST and washed twice. A drop of 1:50 dilution of the first antibody (anti-P450scc or anti-3ßHSD) in PBST was overlayed upon each section for a 30-min incubation, followed by three washes in PBST (5 min each). The biotinylated second antibody was similarly incubated and washed as above. Finally, detection of the antigen was either based on strepavidin-alkaline phosphatase conjugate using ELF kit, or based on ExtrAvidin-peroxidase using DAB for substrate.

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence of mouse P450scc cDNA
In preparation for RT-PCR determinations of the uterine P450scc mRNA levels in mice, we realized that primers designed according to the rat sequence were not necessarily efficient for amplification of the mouse cDNA. Therefore, we cloned the full-length mouse P450scc cDNA and sequenced it. Mouse cDNA was recovered by a 3' RACE approach. The 5' primer was designed according to a previously published promoter sequence of the mouse P450scc gene, including the 5' untranslated region (UTR) of the mRNA (35). Figure 1Go 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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  		Figure 1. Sequence of the mouse P450scc cDNA. Nucleotide sequence (5' to 3' in the mRNA sense) of the full-length mouse P450scc cDNA was obtained and sequenced as described in Materials and Methods. The deduced amino acids are indicated below the corresponding nucleotide sequence. Three asterisks denote the stop codon. Nucleotide number one denotes transcription start site, as deduced from the rat P450scc cDNA. The coding region extends from nucleotide 43 to 1623. Putative polyadenylation signal is underlined.

 
Uterine P450scc, 3ßHSD VI, and P450c17 mRNAs during early pregnancy
According to earlier studies, the three key steroidogenic enzymes regulating placental steroidogenesis in rodents are P450scc, 3ßHSD VI and P450c17 (11, 24, 27, 28, 31, 32, 46). To characterize the expression profile of these genes throughout the earlier stages of mouse pregnancy, we analyzed the levels of their mRNAs by a semiquantitative RT-PCR approach. Total RNA was prepared from the whole uterine tissue or isolated implantation sites on each day of pregnancy. RT-PCR analyses of these extracts showed significant expression of P450scc commencing at the time of implantation of the blastocyst on day 4.5 post coitum (Fig. 2AGo). 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. 2CGo), 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. 2DGo). Unlike P450scc and 3ßHSD VI, which were expressed in the decidual cells of E9.5 sites (Fig. 2BGo), 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. 2EGo). 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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  		Figure 2. Expression of uterine P450scc, 3ßHSD VI, and P450c17 mRNA during early pregnancy. Total RNA was extracted from the following uterine tissues, prepared as described in Materials and Methods: whole uterine horns of pregnant mice on days 3.5–5.5; myometrium-free implantation sites from days 6.5–10.5; extracts of E8.5-E10.5 sites excluding the embryo. Reverse-transcriptase reaction (200 ng RNA samples) and further PCR were conducted as described in Materials and Methods. A, Autoradiogram depicting the PCR fragments obtained for three pairs of primers amplifying P450scc (536 bp), 3ßHSD VI (405 bp) and L19 (194 bp) in each sample lane. The level of the ribosomal protein L19 (L19) PCR product, which does not change throughout pregnancy, served for quantitative normalization of the steroidogenic gene products. B, Similar RT-PCR analysis was performed using RNA extracts from isolated giant trophoblast cells, decidual tissue, or whole implantation site of E9.5 uterus. C, Phosphorimager quantitation of data shown in panels A and D. Data are representative of two independent experiments repeated with similar results. D, Sets of P450c17 and L19 primers were used to generate RT-PCR fragments (361 and 194 bp, respectively) representing the profile of P450c17 during pregnancy, as described in panel A. E14.5 Pl., 14.5 day p.c. placental tissue. E, RT-PCR analysis of P450c17 mRNA levels in mechanically separated uterine elements as described in Panel B.

 
Expression of P450scc protein during early-mid pregnancy
To determine the level of P450scc protein during early pregnancy we performed Western blot analysis using protein extracts prepared from intact uteri and isolated implantation sites (Fig. 3AGo). 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. 3AGo). 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. 3BGo). 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. 3AGo), in accordance with a similar pattern observed for the mRNA profile.



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  		Figure 3. Expression of P450scc protein during early pregnancy. A, Protein extracts of uterine tissue and isolated implantation sites were prepared through days 5.5–11.5 as described in Materials and Methods. Chemiluminescence-based Western blot analysis was performed using 25 µg protein. Ovarian corpora lutea extract (12.5 µg) served for control P450scc. B, Isolated decidual cells of E6.5 were metabolically labeled with 35S-Methionine and P450scc was immunoprecipitated as described in Materials and Methods. Left lane, Decidual cells; center lane, FSH-induced granulosa cells; right lane, control granulosa cells without FSH treatment. C, Low power immunohistochemical staining of an E9.5 implantation site sectioned sagittal-to-embryo/transverse-to-uterus. Artifact staining is seen in blood vessels and blood sinuses (bv and bs, respectively). G, Trophoblast giant cells; db, decidua basalis; dc, decidua capsularis; mes, mesometrium; s, stroma; m, myometrium; p, placenta; yc, yolk cavity; e, embryo. Bar, 1 mm. D, Depicts the antimesometrial area boxed in panel C. Note the strong cytoplasmic labeling of the trophoblast giant cells. The decidua capsularis (dc), the stroma (s) and the myometrium are not labeled. ys, yolk sac; ery, primitive erythrocytes; mry, maternal erythrocytes. Bar, 25 µm.

 
To localize P450scc in specific cell types of the pregnant uterus, tissue sections were prepared using different approaches for immunohistochemical analyses. We failed to demonstrate significant P450scc staining in the decidua cells of early pregnant uteri, probably due to low expression of the antigen, in addition to high background staining that we could not reduce. Transvere sections of E9.5 uteri showed substantial P450scc staining specifically localized in the giant trophoblast cells surrounding the embryonal cavity (Fig. 3Go, C–D). No immunostaining for P450scc protein was detected in E9.5 decidua capsularis or decidua basalis (Fig. 3DGo).

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. 4AGo). 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. 4FGo), whereas bright mitochondrial P450scc immunofluorescent signals were typical for FSH-treated cells (Fig. 4GGo). Due to the relative short exposure to FSH (16 h), some of the granulosa cells were less responsive and expressed less P450scc (Fig. 4GGo), similarly to the immunoreactive enzyme levels observed in the decidual cells (Fig. 4AGo).



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  		Figure 4. Immunolocalization of P450scc in decidua and giant trophoblast cells by fluorescence and electron microscopy. A, Decidual cells of day 6.5 show faint immunofluorescence staining of mitochondrial P450scc. Bar, 20 µm. B, Anti-P450scc immuno-gold labeling of thin electron microscope section depicting 6.5-day decidual cells. Most of the gold particles (18 nm, arrowheads) are specifically localized in the mitochondria (m). Note the lamellar and plated cristae structures (arrows) of the mitochondria. Bar, 350 nm. C, Phase-contrast micrograph of E9.5 giant trophoblast cell culture depicted 48 h after seeding. Note the giant nuclei (n) and numerous dark lipid droplets surrounding the nucleus. This culture was used for immunocytochemical staining, as shown in panel D. Bar, 100 µm. D, a single giant cell immunostained with P450scc antiserum. Note ample labeling of small mitochondria (arrows), the majority of which are stacked in the brightly stained perinuclear space. n, Nucleus. Bar, 30 µm. E, Anti-P450scc immuno-gold labeling of a thin electron microscope section depicting a 9.5-day giant cell. Heavy gold particle labeling is observed in small and round mitochondria (m). The cristae ultrastructure is mostly vesicular (round white areas over gray background consisting of the mitochondrial matrix). cyt, Cytosol; L, cholesterol lipid droplets. Bar, 200 nm. F, Granulosa cells were cultured for 16 h before fixation and immunofluorescence staining with P450scc antiserum, as described in panel A. Note lack of mitochondrial signals. n, Nucleus. Bar, 25 µm. G, Granulosa cells were treated with FSH (100 ng/ml) for 16 h before further processing for immunofluorescence staining with P450scc antiserum. Note strong mitochondrial signals typical of most of the cells. Arrows denote faint mitochondrial signals in some of the cells (*) expressing less P450scc.

 
Electron microscopy studies revealed a low, but significant and specifically localized immuno-gold labeling of P450scc in the decidual mitochondria (Fig. 4BGo). The low gold labeling of the decidual mitochondria is consistent with the lamellar formation of the cristae (Fig. 4BGo), typical of an early-differentiating steroidogenic cell (33). By contrast, the highly steroidogenic mitochondria of the E9.5 giant trophoblast cells (Fig. 4Go, C and D) were heavily decorated by immuno-gold particles in the vesicular structures of the mitochondrial cristae (Fig. 4DGo). 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. 2Go), 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 5Go, 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. 5Go, D–E. Clearly, 3ßHSD VI protein was not expressed in these migrating cells observed on day 7.5 (Fig. 5EGo). However, by E9.5, the giant trophoblast cells exhibited an enormously high accumulation of the enzyme (Fig. 6BGo). 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. 6Go, B–D).



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  		Figure 5. Localization of 3ßHSD type VI mRNA and protein in sections of E6.5 and E7.5 implantation sites. A, an E6.5 histological micrograph of a sagittal-to-embryo/transverse-to-uterus orientation section (hematoxilin-eosin stain). e, Embryo; dc, decidua capsularis; db, decidua basalis; s, stroma; uc, residual uterine cavity; bs, blood sinus; m, myometrium; mes, mesometrium. Bar, 600 µm. B, Fluorescence in situ hybridization was performed using a consecutive section of the implantation site shown in panel A (box B). Note the expression of 3ßHSD VI mRNA in the decidual cells (dc), but not in the embryo (e) or the stroma (s). Bar, 100 µm. C, Higher magnification of the boxed area in panel B. As expected, the labeling is specifically restricted to the cytosol and excluded from the nuclei (n). Bar, 20 µm. D, ELF-immunofluorescence staining (see Materials and Methods) of 3ßHSD VI expressed in a E7.5 section viewed from the antimesometrial side of the embryo. Note ample expression of 3ßHSD VI protein in the decidua capsularis (dc). By contrast, 3ßHSD VI protein is not observed in the embryo (e), the degenerating old uterine epithelium (due), or migrating giant trophoblast cells (arrowheads). Bar, 45 µm. E, Lack of 3ßHSD VI protein in the migrating giant trophoblast cells (arrowhead). n, Giant cell nucleus; e, embryo; due, degenerating old uterine epithelium; dc, heavily stained decidua capsularis cells. Bar, 20 µm.

 


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  		Figure 6. Expression of 3ßHSD VI protein during day 9.5 of gestation. A, Depicts an E9.5 histological micrograph of a sagittal-to-embryo/transverse-to-uterus orientation section (hematoxilin-eosin stain). e, Embryo; ys, yolk sac; dc, decidua capsularis; db, decidua basalis; al, allantois; pl, placenta; dc, decidua basalis; m, myometrium; mes, mesometrium. Bar, 500 µm B, box B in panel A depicts a similar area in a consecutive section prepared for ELF-immunofluorescence. Note the extremely high levels of 3ßHSD VI expressed in the giant trophoblast cells (arrowheads). None of the following cell types expressed the enzyme: allantois (al), placenta (p), decidua capsularis (dc). decidua basalis (db) and the yolk sac (ys). Bar, 120 µm. C, box C in panel A depicts a similar area in a consecutive section. Note lack of 3ßHSD VI expression in the embryo (e). Bar, 120 µm. D, A higher magnification of the uterine wall as depicted in box D of panel A. Note the expression of 3ßHSD VI in the two-layered giant cells, but none in the decidua capsularis (dc), the stroma (s) and the two layers of the myometrium (m, arrows). Bar, 100 µm. E, Histological micrograph of an E14.5 uterus sectioned and stained similarly to A. e, Embryo; ys, yolk sac; lb, labyrinth; sp, spongiotrophoblast; db, decidua basalis; mes, mesometrium. Bar, 2 mm. F, box F in panel E depicts a similar area in a consecutive section prepared for immunohistochemical detection of 3ßHSD. Bar, 100 µm. G, Higher magnification of box G in panel E. lb, labyrinth; arrows depict remnants of giant trophoblast cells. Bar, 25 µm.

 
Assays of 3ßHSD activity determined in monolayers of isolated decidua cell dispersates (day 6.5 p.c.) showed that these cells convert 3H-pregnenolone to 3H-progesterone (Fig. 7Go). 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. 8Go) 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. 6Go, 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. 8Go) and the enzyme was no longer expressed in the mature placenta on day 14.5 (Fig. 6Go, 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. 8Go).



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  		Figure 8. Expression of 3ßHSD VI in mid-term placenta. A, Western blot analysis (50 µg protein/lane) of P450scc, StAR and 3ßHSD VI in E9.5-E13.5 placenta. Giant trophoblast cells including the placenta and allantois were mechanically separated from E9.5 uteri. Placenta were collected at the indicated day of gestation. Lane 1 depicts the ovarian type I of 3ßHSD, which migrates faster in SDS-PAGE (32 ).

 
Steroidogenesis accessory proteins
We also examined the expression of three accessory proteins that are indispensable for pregnenolone production in the gonads and cortical cells of the adrenal, i.e. ferredoxin, ferredoxin reductase (47), and StAR. RT-PCR analysis shows that, at its prime developmental stage, the E7.5 decidua tissue expresses P450scc, StAR, ferredoxin and ferredoxin-reductase transcripts (Fig. 9Go, 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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  		Figure 9. Expression of steroidogenic accessory proteins. Total RNA was prepared for RT-PCR analysis from the following uterine tissues, as described in Materials and Methods: whole uterine horns of pregnant mice on day 3.5 p.c.; myometrium-free implantation sites from day 7.5 p.c. (lane 2); three different RNA samples were prepared from E9.5 implantation sites, including embryo, giant trophoblast cells and decidua. In addition, RNA from the ovaries of the pregnant mice was used for positive control (lane 6). RT-PCR analyses were performed (100 ng/reaction) as described in Materials and Methods. The autoradiograms present the following PCR products, which were obtained in the presence of the following primers: P450scc (536 bp) and StAR (246 bp), ferredoxin (Ferr; 289 bp), ferredoxin reductase (Ferr-Red; 567 bp). The level of the ribosomal protein L19 (L19) PCR product (194 bp), which does not change throughout pregnancy (Fig. 2Go and this figure), served as internal standard.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous report we showed that the onset of implantation in mice is associated with the expression of P450scc mRNA in the decidualizing tissue at the anitmesometrial side of the uterine wall (31). By use of in situ hybridization, it was also shown that in addition to P450scc expression in the decidua, this transcript becomes highly abundant in the embryonic giant trophoblast cells, which surround the entire embryonic cavity by days 8.5–10.5 of mouse gestation (31). The unexpected maternal expression of P450scc in the decidua raised the question of whether this tissue is capable of expressing 3ßHSD which is required for progesterone biosynthesis (1). We have previously reported the cloning of a new murine 3ßHSD isoform, 3ßHSD VI (32). This isoform functions as a very efficient NAD+-dependent dehydrogenase/isomerase catalyzing the conversion of pregnenolone to progesterone (32). RT-PCR studies have also shown that 3ßHSD VI mRNA is the only isoform expressed in the uterine tissues between days 4.5- 9.5 p.c., but as expected, this transcript is absent in the ovary or adrenal gland (32).

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.


   Acknowledgments
 
We are grateful to Dr. Ian Mason for providing the 3ßHSD antiserum, Drs. K. H. Hales and D. B. Hales for providing the anti-StAR serum and Mrs. Naomi Kingsley for editorial help.


   Footnotes
 
1 This work was supported by The United States-Israel Binational Sciences Foundation Grant 95–00350 (to J. O.) and NIH U54 HD-31398 (to A. H. P.). Back

Received April 26, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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