Molecular Human Reproduction, Vol. 5, No. 3, 277-286,
March 1999
© 1999 European Society of Human Reproduction and Embryology
Temporal expression of inducible nitric oxide synthase in mouse and human placenta
1 Wellcome Research Laboratories, Langley Court, Beckenham, Kent, BR3 3BS, UK, 2 Department of Anatomy and 3,4 Department of Obstetrics and Gynecology, University of Iowa College of Medicine, Iowa City, IA 52242, USA
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The aim of this study is to investigate the changes in expression and activity of inducible nitric oxide synthase (iNOS) in the developing murine embryo and mouse and human placenta. Using reverse transcription–polymerase chain reaction (RT–PCR), Northern blotting, and in-situ hybridization (ISH) we identified iNOS mRNA in mouse placenta at 9.5, 12, 14, 16, 18 and 20 days post coitum. Northern blot analysis demonstrated that the quantity of murine iNOS transcript was expressed at a stable level between days 12–20 although the level of calcium-independent NOS activity declined with advancing gestation. RT–PCR detected iNOS-specific mRNA in murine embryonic stem cells, but not in embryos at later stages (4-cell or blastocyst). ISH failed to show iNOS-specific mRNA in either murine placenta or the underlying myometrium on day 7, but did so in the trophoblast by day 9.5. Later in gestation, extensive labelling was observed in both spongiotrophoblast and trophoblast giant cells. iNOS mRNA was also detected both in immature human placentae (16–18 weeks) and at term, predominantly in syncytiotrophoblasts and placental artery smooth muscle. In conclusion, iNOS is constitutively expressed in mouse and human placenta at a time and in a location that suggests a role in placentation.
EDRF/embryo/implantation/nitric oxide/trophoblast
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Nitric oxide (NO) is produced from L-arginine by a family of enzymes, the NO synthases (NOS) (Moncada and Higgs, 1993
; Knowles and Moncada, 1994; Mayer and Hemmens, 1997). Three distinct genes encoding mammalian NOS isoenzymes have been identified and are historically classified by their dependence for activity on the level of intracellular calcium. Both endothelial NOS (eNOS or NOS III) and neuronal NOS (nNOS or NOS I) are highly regulated by calcium and calmodulin. The NO generated by these calcium-dependent enzymes has numerous roles in both the cardiovascular and central and peripheral nervous systems. The third isoenzyme, (iNOS or NOS II) binds calmodulin tightly and is thus relatively calcium-independent. It is transcriptionally regulated by cytokines and bacterial endo- and exotoxins and has been implicated in anti-microbial and anti-tumour defences. Another classification of the NOS isoenzymes divides them in constitutive (eNOS and nNOS) and inducible (iNOS). However, recent findings suggest that eNOS and nNOS expression can also be induced and that in some tissues iNOS appears to be present at all times.
The biosynthesis of NO is significantly increased by pregnancy (Weiner et al., 1995). Both plasma and urinary nitrate are elevated in the pregnant human and rat (Conrad et al., 1993a), and increases in NOS activity occur in several tissues of pregnant and oestradiol-treated guinea pigs (Weiner et al., 1994). Thus, the elevated concentrations of circulating nitrate are likely to be a consequence of both the increased production of NO by the maternal peripheral tissues and by the fetus and/or placenta. A gestational-dependent up-regulation in NO synthesis was reported in the rat (Buhimschi et al., 1996) but not human myometrium (Buhimschi et al., 1995). Increased NO production in the rat uterus during pregnancy is a consequence of an increased expression of iNOS (Ali et al, 1997; Buhimschi et al., 1997) and has been localized to decidua, metrial glands, and a minority of uterine myocytes subjacent to the placental attachment site (Sladek et al., 1998).
NOS isoforms are also actively expressed in syncytiotrophoblast (Conrad et al., 1993b; Myatt et al., 1993a, 1997; Buttery et al., 1994; Garvey et al., 1994; Eis et al, 1995). NOS activity in the placental vasculature is calcium-dependent, inhibited by an antagonist of calmodulin, and reactive with a monoclonal antibody raised against bovine eNOS (Myatt et al., 1993b). Northern blotting and sequence analysis of cDNAs identified eNOS in human placenta and showed that the placental eNOS is identical to the enzyme synthesized by human umbilical vein endothelial cells in culture (Garvey et al., 1994).
Various investigators have sought to determine the cellular localization of NOS isoforms in mammalian placenta. Springall et al. (1992) demonstrated histochemically an isoform of NOS in the human umbilical vein and the syncytiotrophoblasts of term placenta using an antibody raised against purified rat brain nNOS. This isoenzyme was subsequently demonstrated by using selective antibodies (Buttery et al., 1994; Garvey et al., 1994) and in-situ hybridization (ISH) (Conrad et al., 1993b) to be eNOS. Using immunohistochemistry, Eis et al. (1995) identified eNOS in the syncytiotrophoblast of first trimester human villous tissue. They found that differentiation of cytotrophoblast into syncytiotrophoblast is correlated with the expression of eNOS. Finally, nNOS was demonstrated by Graf et al. (1994) in the smooth muscle-like cells of the extravascular contractile system of the human placenta where it may modulate the tone of the perivascular contractile sheaths.
Calcium-independent NOS activity was reported by Kukor and Tóth (1994) and Tóth et al. (1995) in the early first trimester human placenta. In an earlier study, we failed to detect iNOS-specific mRNA in human placenta from normal term pregnancies (Garvey et al., 1994; S.Baylis, unpublished data). Gestational age may be relevant to the expression of the placental NOS isoenzymes. Recently, Purcell et al. (1997) reported that iNOS is expressed in rat placenta during late gestation in the peripheral placental layer at the maternal–fetal interface and its expression is down-regulated prior to term, postulating that NO plays a paracrine role in regulating myometrial contractility. However, a decrease in NOS enzymes does not seem to occur at least immediatly before the onset of parturition in humans (Thomson et al., 1997).
Previously, we have found that pregnancy dramatically increased myometrial arginase activity (Weiner et al., 1996). The activity is greatest at the placental implantation site. Since the maternal–fetal interface is rich in a variety of cytokines (Athanassakis-Vassilidis, 1993; Delassus et al., 1994), and since arginase is co-induced with iNOS in macrophage (Corraliza et al., 1995), we postulate that the cytokines present at the placental–maternal interface also stimulate the transcription of iNOS. Using a variety of approaches, we aim to investigate the expression of iNOS-specific mRNA and calcium-independent NOS activity in murine stem cells, embyos and murine and human placenta at specific times.
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Animals and tissues
These investigations were approved by the appropriate Institutional Committees for Human Investigation and Animal Welfare. For polymerase chain reaction (PCR) experiments, placentae from NIH Swiss and BALB/c mice, killed by cervical disclocation, were dissected from each animal at 12, 14, 16, 18 and 20 days post coitum (day 21 = expected day of delivery). At least three animals were studied for each gestational age. Three placentae were pooled from each animal and immediately snap-frozen in liquid nitrogen and stored at –70°C as one sample. Selection of placentae was performed at random and samples were collected in duplicate for each animal (for mRNA extraction and measurements of NOS activity respectively). In a separate experiment, mice were injected i.p. with 25 µg lipopolysaccharide (LPS: Salmonella typhosa) (Difco, Detroit, MI, USA). The spleens were collected 6 h after injection, snap-frozen and stored at –70°C.
For ISH, placentae and embryos from NIH Swiss and BALB/c mice were collected on days 9.5, 14 and 19 of gestation. Briefly, animals were anaesthetized with 5 mg ketamine (Ketalar; Parke-Davis, Morris Plains, NJ, USA) and perfused intracardially with phosphate-buffered saline (PBS) containing 4% paraformaldehyde (pH 7.5). After collection, the tissues were maintained in fixative for 24 h at 4°C. Thereafter, the tissues were washed in PBS, dehydrated through a graded series of ethanol, cleared in xylene and embedded in paraffin. The blocks were stored at 4°C before further processing.
Murine 4-cell and blastocyst-stage embryos were a gift from Roger Williamson, Department of Obstetrics and Gynecology, University of Iowa College of Medicine. J774 murine macrophages from BALB/c female mice were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).
Human placentae at 16–18 weeks gestation were obtained from four healthy pregnant women at the time of elective surgical pregnancy termination. After collection, the samples were immediately immersed in the fixative solution and processed for ISH as described above.
RNA isolation and nucleic acid manipulation
Poly (A)+ RNA was isolated using a FastTrack Kit (Invitrogen, Carlsbad, CA, USA) from murine placentae and J774 macrophages (untreated or activated for 8 h with 10 IU/ml of recombinant murine interferon-
(Difco, Detroit, MI, USA) and 10 µg/ml LPS). Poly (A)+ RNA from term human placenta was obtained from a commercial source (Clontech, Palo Alto, CA, USA). Total RNA from murine embryonic stem cells was a gift from Austin Smith, the Center for Genome Research, University of Edinburgh, UK.
Reverse transcription–polymerase chain reaction (RT–PCR) was performed using the GeneAmp RNA PCR kit (Perkin Elmer, Norwalk, CT, USA) in accordance with the manufacturer's instructions. To control for contamination with genomic DNA, incubations without reverse transcriptase were performed from the same RNA samples.
Murine iNOS was amplified using the specific primers: 5'-GCCTCATGCCATTGAGTTCATCAACC (sense, bases 402–427 of the coding sequence) and 5'-GAGCTGTGAATTCCAGAGCCTGAAG (antisense, bases 750–774 of the coding sequence), to amplify a 372 bp product. Amplification was performed in the presence of 1 mM MgCl2 and used a hot start protocol with 35 cycles (Biometra TRIO-Thermoblock; Biometra, Tampa, FL, USA) of the following sequential steps: 96°C for 35 s, 62°C for 2 min, 72°C for 2 min.
Murine eNOS and ß-actin were also amplified using primers for their human homologues: The eNOS primers corresponded to bases 2864–2888 (5'-TCACTGTAGCTGTGCTGGCATACAG, sense) and 3333–3357 (5'-CATGGTAACATCGCCGCAGACAAAC, antisense) of the coding sequence of human eNOS and bases 554–583 (5'-TGACTGACTACCTCATGAAGATCCTCACCG, sense) and 837–862 (5'-CCACGTCACACTTCATGATGGAGTTG, antisense) of the coding sequence of human ß-actin. These primers generated 493 and 304 bp products, respectively. The amplification was performed as described before except primer annealing was conducted at 58°C for the eNOS primers and 60°C for the ß-actin primers.
The human NOS isoforms were amplified using a multiplex PCR technique. Human eNOS was amplified using the following primers: 5'-CAGTGTCCAACATGCTGCTGGAAATTG (sense, bases 1004–1030 of the coding sequence) and 5'-TAAAGGTCTTCTTCCTGGTGATGCC (antisense, bases 1465–1489 of the coding sequence) and yielded a 485 bp product. The human iNOS primers (5'-CAGTACGTTTGGCAATGGAGACTGC [sense, bases 1770–1794 of the coding sequence] and 5'-GGTCACATTGGAGGTGTAGAGCTTG [antisense, bases 2184–2109]) amplified a 340 bp product. Human nNOS primers (5'-TAGCTTCCAGAGTGACAAAGTGACC [sense, bases 1929–1953 of the coding sequence] and 5'-TGTTCCAGGGATCAGGCTGGTATTC [antisense, bases 2125–2149 of the coding sequence]) amplified a 220 bp product. PCR was performed as for mouse iNOS. Products were visualized on a 2% agarose gel using ethidium bromide staining. To confirm the identity of the NOS PCR products, the bands with the expected size were either sequenced directly using an ABI 373 sequencer (University of Iowa core DNA facility, Iowa City, IA, USA) or cloned into the vector pT7 Blue (Novagen, Madison, WI, USA) prior to DNA sequencing. Restriction enzyme digestion was also used to confirm the authenticity of the PCR products obtained through mutiplex PCR amplification.
Northern hybridization
Poly (A)+ placental RNA (1.25 µg) from each animal and from J774 macrophages (positive control) was fractionated through formaldehyde/agarose gels and transferred to Hybond-N (Amersham, Arlington Heights, IL, USA). The RNA was fixed to the filters by UV radiation and the filters hybridized overnight at 65°C in 7% sodium dodecyl sulphate (SDS)/0.5 M NaPO4 /1% bovine serum albumin (BSA) (Church and Gilbert, 1984) with the cloned 372 bp murine iNOS PCR product labelled by random priming. After hybridization, the filters were washed at 65°C with 0.1x sodium chloride/sodium citrate (SSC) (0.15 M NaCl and 0.015 M NaCitrate) containing 0.1% SDS. The filters were then re-probed for ß-actin to normalize for possible RNA loading differences.
In-situ hybridization
The sections for study were both generated in our laboratory according to established methodology or purchased (Novagen, Madison, WI, USA). Histological sections (5 µm) were cut in random orientation at days 8–10 gestation, and in sagittal orientation at gestation day 14 and older. The sections were mounted onto Plus X-treated glass slides (Fisher, St Louis, MO, USA) and stored at 4°C. The sections were de-waxed in xylene and rehydrated through a series of graded ethanol solutions immediately prior to ISH.
Oligonucleotide probes
The probes were prepared as synthetic oligonucleotides complementary to the iNOS cDNA. A three probe cocktail, complementary to nucleotide bases 445–480, 1257–1292 and 1842–1879 of the coding sequence of the murine iNOS cDNA was used throughout the study. The oligonucleotides were labelled with [
-35S]-dATP (Amersham) using terminal deoxynucleotidyl transferase (Gibco-BRL) in a total volume of 50 µl consisting of 100 ng oligonucleotide in 27 µl DEPC-treated water, 10 µl tailing buffer (Gibco-BRL), 1 µl BSA (2%) and 6.5 µl terminal deoxynucleotidyl transferase (15 IU µl), as described previously (Pratt et al., 1993). Following incubation at 37°C for 8 min., the labelling reaction was stopped by addition of 5 µl EDTA (500 mM; Sigma, St Louis, MO, USA) and denatured at 70°C for 10 min. The probes were then purified by centrifugation through a Biogel P30 chromatography column and stabilized in 40 mM dithiothreitol (DTT).
ISH histochemistry
Tissue sections were hybridized as described previously (Pratt et al., 1993). Briefly, sections were incubated with 50 µl/cm2 of hybridization solution consisting of dextran sulphate (10%), Denhardt's solution (1x), DTT (0.1M), tRNA (0.25 mg/ml), polyadenylic acid (0.25 mg/ml), denatured herring sperm DNA (0.25 mg/ml), deionized formamide (50%) and [-35S]-dATP-labelled oligonucleotide probes (300 000 c.p.m./50 µl per probe). The sections were covered with Sealon-film (Fuji Film, Tokyo, Japan) and incubated in a sealed moist chamber at 43°C for 18–20 h. Thereafter, the Sealon-film was floated off in 1x SSC at room temperature after which the sections were washed twice in 1x SSC at 55°C, twice in 0.5x SSC at 55°C and once in 0.5x SSC at room temperature. DTT (10 mM) was included in all SSC washing steps. Subsequently, the sections were dipped in diethyl pyrocarbonate-treated water, dehydrated through ethanol and autoradiographed using ß-max Hyperfilm (Amersham) for 3–4 weeks. The films were developed and fixed in D19 and Unifix (Eastman Kodak, Rochester, NY, USA) respectively. Cellular resolution of the hybridization signal was obtained by dipping of sections into LM-1 Hypercoat emulsion (Amersham) and exposing for 6–8 weeks at 4°C. After developing and fixing, the sections were counterstained with Cresyl Violet (0.1%). To control for non-specific hybridization, a 100-fold excess of unlabelled oligonucleotide probe was added to the hybridization solution. In some series, an additional control employed RNase A treatment (100 µg/ml). These procedures completely abolished hybridization signals.
Assays for NOS activity
NOS activity was determined by measuring the conversion of L-[U-14C] arginine to [U-14C] citrulline (Salter et al., 1991). The tissue was rapidly removed and frozen in liquid nitrogen. The stored placental samples were homogenized at 0°C in a buffer (pH 7.0 at 20°C) containing 320 mM sucrose, 50 mM Tris, 1 mM EDTA, 1 mM DTT, phenylmethylsulfonyl fluoride (100 µg/ml), leupeptin (10 µg/ml), soybean trypsin inhibitor (100 µg/ml), and aprotinin (2 µg/ml). The crude homogenates were centrifuged at 0°C at 12 000 g for 20 min., the pellet discarded and the post-mitochondrial supernatants placed on ice. NOS activity was determined within 1 h of preparation by measuring, in duplicate the conversion of L-[U-14C] arginine to L-[U-14C] citrulline. L-Valine (50 mM) was added to the reaction buffer to minimize any interference from arginase. Total activity was the difference between untreated samples and samples containing both 1 mM EGTA and 1 mM N
-monomethyl-L-arginine. Calcium-independent activity was the difference between untreated samples and the samples containing 1 mM EGTA. Calcium-dependent activity was calculated by subtracting calcium-independent activity from total activity.
Statistical analysis
The results are presented as mean +1 SEM. Multiple comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by multiple post-hoc Tukey tests. P < 0.05 was considered to be statistically significant.
Macrophage and placental alkaline phosphatase-specific staining
Histological sections from the same tissue blocks subjected to ISH were deparaffinized and then incubated for 1 h at room temperature with either a rabbit polyclonal antibody for placental alkaline phosphatase (1:100) or a mouse monoclonal antibody for macrophage CD68 (Dako, Carpinteria, CA, USA) (1:250). The sections were washed three times for 10 min in PBS and incubated with either a mouse anti-rabbit antibody or rabbit anti-mouse antibody respectively, conjugated to horseradish peroxidase. The sections were again washed three times in PBS and then incubated in a solution of hydrogen peroxide plus diaminobenzidine to produce the horseradish peroxidase-catalysed reaction product. The sections were again washed, lightly counterstained with haematoxylin, mounted and then photographed. Control slides had mouse non-immune serum (Sigma) substituted for the primary antibody.
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Presence of iNOS-specific mRNA by RT-PCR and Northern blot
iNOS-specific cDNA was amplified from the developing mouse placentae on days 12, 14, 16, 18 and 20 post coitum (Figure 1A
). Direct DNA sequence analysis of the PCR products confirmed they were identical to the published sequence for the murine macrophage cDNA (Lyons et al., 1992). Positive controls consisted of mRNA from J774 murine macrophages treated with interferon- and bacterial LPS. While a small amount of iNOS mRNA was detected by PCR in untreated J774 cells, it was undetectable by Northern blotting even after prolonged exposure (data not shown). The mRNA for eNOS was also detected by RNA–PCR in the placenta at all the times examined (Figure 1B). ß-Actin expression was used as a control for loading and RNA integrity of all samples (Figure 1C).
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Northern blot analysis of murine placental mRNA revealed that the quantity of the 4 kb iNOS transcript remained largely unchanged between days 12 and 20 of pregnancy (Figure 2
). The quantity of placental iNOS message present was less than that observed in J774 murine macrophages treated with interferon- and bacterial LPS. Probing for ß-actin confirmed the loading of approximately equivalent amounts of mRNA.
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Although we previously reported that eNOS was the only NOS isoform present in human placenta at term based on Northern blot studies (Garvey et al., 1994
), we now find that iNOS-specific mRNA is also present in the human placenta at term using a multiplex PCR assay and conditions modified to enhance sensitivity (Figure 3). The authenticity of these PCR products was confirmed by restriction enzyme digestion. nNOS (expected size of 220 bp) was not detected in any of the samples analysed.
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iNOS-specific mRNA was also identified by RT–PCR in murine embryonic stem cells (derived from the inner cell mass of the blastocyst) (Figure 4
) but not the 2-cell embryo or the whole blastocyst embryo (not shown).
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Placental NOS activity
Calcium-dependent NOS activity of murine placenta did not change with advancing gestational age, averaging 1.79 pmol/min/mg protein. In contrast, the calcium-independent NOS activity decreased with time from days 14–20 gestation (day of delivery). A statistically significant decrease was observed by day 18. However, earlier in gestation (day 12) calcium-independent NOS activity was not measurable (Figure 5
). The level of calcium-independent NOS specific activity from day 14 post-coitum placental tissue was similar to that found in the spleens of mice treated with LPS.
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ISH studies
iNOS mRNA was not seen in either the developing placenta or the underlying myometrium of the day 7 mouse (Figure 6A
). It was found by day 9.5 post coitum where there was extensive staining for iNOS in the spongiotrophoblast and trophoblast giant cells (Figure 6B and C). Subsequently (days 14 and 19), staining was observed in both uterine epithelium and stroma (Figure 7A–D). Human placenta at weeks 16–18 also contained iNOS mRNA, but predominantly within the syncitiotrophoblast and less in the cytotrophoblastic cells (Figure 8A and B). In addition, extensive iNOS-specific staining was observed in the placental arteriolar smooth muscle (Figure 8C and D).
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Serial sections from human placenta that were probed for iNOS were either stained for the presence of macrophages (Figure 9A
) or for alkaline phosphatase activity (Figure 9B). Macrophages were found both within and outside the microvasculature and in the cytotrophoblast. They clearly did not coincide with the iNOS mRNA in either the trophoblast or vascular smooth muscle.
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This study demonstrates for the first time that: (i) iNOS-specific mRNA is present in embryonic stem cells derived from the inner cell mass of the blastocyst but not detectable in the two cell embryos, whole blastocysts and mouse placenta on day 6 but becomes present by day 9.5 post coitum; (ii) iNOS gene is expressed in both spongiotrophoblast and trophoblast giant cells of the developing and mature murine placenta; (iii) although the levels of iNOS mRNA appear to be constant from days 12 to 20, calcium-independent NOS activity becomes detectable around day 12, is maximal on day 14, and falls with advancing gestation; and (iv) iNOS mRNA is also present in human placenta during midtrimester within the wall of the placental vessels and predominantly in syncytio- but also in cytotrophoblast; it does not co-localize with macrophages.
In 1993, Myatt et al. (1993a) and Conrad et al. (1993b) reported that human placenta at term expresses only the eNOS isoform. Both studies identified a minor calcium-independent NOS activity (only 5–6% of the total NOS activity) without being able to identify iNOS mRNA (Conrad et al., 1993b) or protein (Myatt et al., 1993b). However, recently, Myatt et al. (1997) showed that iNOS immunostaining could be detected in human term placenta mainly in the villous tissue and the Hofbauer cells of the villous stroma, and less in the syncytiotrophoblast and vascular endothelium (Myatt et al., 1997). This is in contrast to another recent report that identified iNOS mRNA only in term placentae from pregnancies complicated with gestational diabetes (Schonfelder et al., 1996). Our present study suggests that iNOS is expressed in the normal developing human placenta and at term. This finding is consistent with Ramsay et al. (1996) who describe significantly higher calcium-independent NOS activity early in gestation than at term. However, our study used two different methods (ISH and PCR) with different sensitivities to detect iNOS mRNA in early and term human placenta respectively. At this time, we have not attempted to quantify gestational changes between these two points. In contrast, we used PCR to detect iNOS mRNA as well as Northern blotting to quantify changes in murine placentae, since both methods taken alone have limitations, e.g. quantitative errors due to plateau effects (for PCR) or low sensitivity (for the Northern blots). Our ISH results in human developing placenta show that iNOS is also expressed in cytotrophoblastic cells. This suggests that iNOS is expressed earlier in placentation than eNOS, since most authors agree that eNOS is found exclusively in the syncytiotrophoblast (Conrad et al., 1993b; Myatt et al., 1993a; Buttery et al., 1994; Eis et al., 1995). Our results are consistent with Eis et al. (1995) who observed NADPH diaphorase activity in the trophoblast of the human first trimester placenta that did not correlate with eNOS expression. It is possible the NADPH diaphorase activity represents iNOS.
Two morphologically and functionally distinct zones are evident in murine placenta (Soares et al., 1996); the junctional zone (peripheral) and the labyrinth zone (central). The labyrinth contains trophoblast giant cells, syncytial trophoblast cell, fetal mesenchyme and vasculature and is the site of fetal–maternal vascular exchange. The junctional zone, contains trophoblast giant cells (in contact with the maternal decidua) and spongiotrophoblasts. The junctional zone is the site of hormonal synthesis, maternal–fetal paracrine signalling and fetal vasculature penetration. Trophoblast giant cells exhibit invasive as well as endocrine activities. Spongiotrophoblasts, which are morphologically distinct from giant cells have endocrine and energy reserve functions by accumulating glycogen (glycogen cells). The level of expression of iNOS mRNA by ISH appears greatest in the junctional zone, where the placenta is most intimately in contact with the maternal surface. This has also been suggested by Purcell et al. (1997) for iNOS protein in rat placenta. Presumably, the concentration of cytokines is also highest here (Delassus et al., 1994; Weiner et al., 1996) which is consistent with the high levels of arginase activity in the myometrium directly underlying the placental bed (Athanassakis-Vassiliadis, 1993), since iNOS and arginase are co-induced in the macrophage (Corraliza et al., 1995).
Although iNOS-specific staining was not seen in macrophages, it was seen within the the vascular smooth muscle of a placental arteriole. This observation parallels the work of Galea et al. (1995) who noted iNOS-specfic mRNA and protein in the brain microvasculature and arterioles of the perinatal rat (Nussler and Billiar, 1993). In our studies, iNOS-specific staining was not seen in the area of the placental capillaries. This is apparently the first demonstration of iNOS expression in vascular smooth muscle from pregnant healthy subjects earlier in pregnancy (16–18 weeks gestation).
Historically, iNOS expression has been considered a sign of disease. A variety of cytokines induce iNOS in macrophages, chondrocytes, vascular smooth muscle, hepatocytes and myocardiocytes (Charles et al., 1993; Geller et al., 1993; Nussler and Billiar, 1993; Tsujino et al., 1994). In many of these scenarios, the NO produced is toxic. However, this paper shows that iNOS expression within the placenta of both mice and humans is part of normal physiology and development. Calcium-independent NOS activity and/or iNOS-specific mRNA have also been reported in the normal bronchiolar epithelium from human adults and fetuses, rat kidney arterioles, human and rat granulosa cells, and in myometrial mast cells and endometrial epithelial cells from the mouse uterus (Kobzik et al., 1993; Ahn et al., 1994; VanVoorhis et al., 1994 VanVoorhis et al., 1995; Huang et al., 1995).
The role of iNOS in placentation, if any, can only be the subject of speculation at this stage. One possibility is that it enhances angiogenesis. Jenkins et al. (1995) transfected a human adenocarcinoma cell line that produced NO at significantly lower levels than activated macrophages. When transplanted into nude mice, the transfected cells were associated with significant amounts of angiogenesis compared with control tumour cells. However, iNOS is not necessarily an absolute requirement for murine development, since the iNOS-deficient mice develop and reproduce in an apparently normal fashion (MacMicking et al., 1995; Wei et al., 1995). Indeed, this is also true of eNOS since eNOS-deficient mice reproduce (Huang et al., 1995b). It is possible that compensatory mechanisms exist in both the eNOS and iNOS-deficient mice which allow them to reproduce normally. For example, nNOS-deficient mice express the eNOS isoenzyme in place of nNOS (O'Dell et al., 1994). The requirement for NO in placental function will perhaps be further elucidated by the generation of double knockout mice deficient in both eNOS and iNOS.
The intriguing discrepancy between gestational changes in iNOS activity in the presence of a constant mRNA expression suggest that post-transcriptional or post-translational modifications may be involved. Such mechanisms have been previously described for iNOS in mouse macrophages (Weisz et al., 1994).
In conclusion, iNOS is expressed in both human and mouse developing and mature placenta. The location of iNOS mRNA suggests a role for iNOS in placentation and embryo development. Furthermore the decrease in NO production from the murine placenta before term may be involved in the preparatory process for delivery in this species.
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Acknowledgments |
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We thank Dr Irina Buhimschi for her editorial assistance, Hugh Spence for oligonucleotide synthesis, Marcus Oxer for DNA sequencing and Dr Scott Nelson, Nick Davies and Barry Warburton for their assistance. The authors would also like to acknowledge Dr Salvador Moncada for his continued support of their research. Sponsored in part by grants from the United States Public Health Service-, HL49041 (CPW), HL51735 (CPW).
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Notes |
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5 To whom correspondence should be addressed at: Perinatal Research Laboratory, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Maryland School of Medicine, 4th Floor 405 West Redwood Street, Baltimore, MD 21201, USA
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Submitted on August 27, 1998; accepted on December 16, 1998.
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