Molecular Human Reproduction, Vol. 6, No. 3, 283-287,
March 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Expression and localization of inducible nitric oxide synthase in human non-pregnant and early pregnant endometrium
Department of Obstetrics and Gynaecology, Faculty of Medicine, Tokyo Medical and Dental University, 1–5–45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Abstract
The aim of the present study was to investigate the expression and distribution patterns of inducible nitric oxide synthase (iNOS) in human non-pregnant and early pregnant endometrium using Northern blot analysis, reverse transcription–polymerase chain reaction (RT–PCR) and immunohistochemistry. Northern blot analysis revealed the expression of iNOS mRNA in human decidua and chorionic villi in the first trimester but not in the endometrium at any stage of the menstrual cycle. Nested RT–PCR, however, detected iNOS mRNA in human endometrium at all stages of the menstrual cycle. Immunohistochemical staining of the secretory endometrium using an anti-human iNOS polyclonal antibody revealed labelling specifically concentrated in glandular epithelial cells. Staining was absent in stromal cells. However, iNOS staining was positive in decidualized stromal cells in tissues obtained in the first trimester of pregnancy. Furthermore, extensive staining was observed in both syncytiotrophoblastic and cytotrophoblastic cells. The finding of a large amount of iNOS mRNA at the feto–maternal interface throughout the first trimester of pregnancy suggests that iNOS may play an important role in the maintenance of pregnancy.
chorionic villi/decidua/endometrium/inducible nitric oxide synthase
Introduction
Nitric oxide (NO) is a highly reactive inorganic free radical with widespread biological effects (Moncada and Higgis, 1993
). It is now recognized that NO is generated by many cell types and is involved in diverse functions including the regulation of vascular tone, neurotransmission and mediation of immune response (Nathan, 1992). This ubiquitous molecule is considered to be an important paracrine messenger controlling vascular constriction and other changes in several physiological and pathophysiological events that occur in endocrine organs.
NO is synthesized by oxidative deamination of the semiessential amino acid L-arginine catalysed by the enzyme, nitric oxide synthase (NOS) (Palmer et al., 1988), which exists in three main isoforms. A constitutive, calcium-dependent form is found in endothelial cells (eNOS) and neurons (nNOS), while a calcium-independent, inducible form (iNOS) is present in macrophages, neutrophils and other cell types (Moncada et al., 1991). The third isoenzyme is transcriptionally regulated by cytokines and bacterial endotoxins and has been implicated in anti-microbial and anti-tumour defence mechanisms. However, recent findings suggest that eNOS and nNOS expressions can also be induced and that in some tissues iNOS appears to be present at all times (Förstermann et al., 1995).
Until now, the expression patterns of NOS isoforms in human endometrium have not been fully elucidated. Recent immunohistochemical studies have revealed the presence of eNOS protein in epithelial and endothelial cells of human endometrium (Telfer et al., 1997; Ota et al., 1998; Tschugguel et al., 1998), and the expression of eNOS mRNA in the endometrium has also been demonstrated using in-situ hybridization (Telfer et al., 1995), Northern blot analysis (Tseng et al., 1996) and reverse transcription–polymerase chain reaction (RT–PCR) (Telfer et al., 1997; Tschugguel et al., 1998). iNOS immunoreactivity was demonstrated in endometrial glandular epithelium (Telfer et al., 1997; Tschugguel et al., 1998), and iNOS mRNA was detected in epithelial cells (Telfer et al., 1997), in stromal cells (Yoshiki et al., 1999) and in the secretory phase endometrium (Tschugguel et al., 1998) by RT–PCR. However, the patterns of iNOS expression in human endometrium with respect to the phases of the menstrual cycle and during the first trimester of pregnancy have not yet been clearly established. On the other hand, a role for NO in the process of implantation and in the maintenance of pregnancy has been proposed from animal studies.
The aim of the present study was to investigate the changes in iNOS expression in human non-pregnant and early pregnant endometrium using Northern blot analysis, RT–PCR and immunohistochemistry.
Materials and methods
Tissue samples
Human endometrial samples were obtained from parous women (n = 15) with regular menstrual cycles undergoing hysterectomy for benign indications at the Tokyo Medical and Dental University Hospital, Japan. The date of the endometrium was histologically assessed according to the standard criteria (Noyes et al., 1950). None of the patients was under hormone therapy. The first trimester decidual and chorionic villous tissues (6–10 weeks of gestation) were collected from women (n = 9) undergoing legal terminations of normal pregnancy by surgical evacuation of the uterus. None of the women received cervical ripening agents (for example prostaglandin analogues and antiprogestins). The decidua and chorionic villi were thoroughly washed with physiological saline and carefully separated by dissection under microscopy. Informed consent was obtained in each case and the study was approved by the local ethics committee. After collection, the samples were snap-frozen in liquid nitrogen and stored at –80°C.
RNA extraction
Total RNA was isolated from frozen tissues by the single step acid guanidinium thiocyanate–phenol–chloroform extraction method (Chomczynski and Sacchi, 1987).
Deoxyribonuclease (DNase) treatment was performed at 37°C for 15 min in a 50 µl reaction mixture containing 20 IU of RQ1 ribonuclease (RNase)-free DNase (Promega, Madison, WI, USA) and 5 µl of 10x incubation buffer (500 mmol/l Tris–HCl, pH 8.0, 10 mmol/l MgCl2, 1 mg/ml bovine serum albumin). After incubation, 150 µl of diethyl pyrocarbonate (DEPC)-treated water was added to the incubation mixture and RNA was extracted with 200 µl of phenol/chloroform, followed by extraction with an equal volume of chloroform. DNase was inactivated by heating to 65°C for 10 min before cDNA synthesis.
Poly(A)+ mRNA was isolated with Oligotex-dT30 (Takara, Otsu, Japan). Quantity and quality of the extracted poly(A)+ mRNA were determined by spectrophotometry at 260 and 280 nm and UV-light visualization of ethidium bromide-stained agarose gels.
Northern blot analysis
Cloned cDNA fragment containing 505 bp of human iNOS-specific sequence was used as the probe for Northern blot analysis. Total RNA (20 µg) and 5 µg of poly(A)+ mRNA were denatured and subjected to electrophoresis in 1% agarose/5.4% formaldehyde gel in 1x MOPS buffer (20 mmol/l MOPS, 50 µmol/l sodium acetate, 1 mmol/l EDTA, pH 7.0). The RNAs were transferred onto a Biodyne A membrane (Pall, Glen Cove, NY, USA) and immobilized by UV cross-linking. Hybridization was carried out with 25 ng of an [
-32P] dCTP-labelled cDNA probe in QuikHyb Hybridization Solution (Stratagene, La Jolla, CA, USA) at 68°C for 1 h. Membranes were washed initially with 2x standard saline citrate (SSC; 1x SSC = 150 mmol/l NaCl, 15 mmol/l sodium citrate)/0.1% sodium dodecyl sulphate (SDS) at room temperature twice for 15 min, followed by washing at increasing stringency up to 0.1x SSC/0.1% SDS at 60°C for 30 min. Autoradiographs were obtained by exposure to X-ray films at –80°C.
PCR–Southern blot analysis
Complementary DNA was prepared from 1 µg of DNase-treated RNA by RT under the following conditions: RNA dissolved in sterile DEPC-treated water was heated to 80°C for 5 min with 50 ng of random hexanucleotide primers (Takara), followed by annealing. After this, the samples were added to a 20 µl reaction mixture containing 50 mmol/l Tris–HCl (pH 8.3), 40 mmol/l KCl, 5 mmol/l MgCl2, 0.5% Tween-20 (v/v), 10 mmol/l dithiothreitol, 1 mmol/l of each dNTP, 20 IU of RNase inhibitor (Boehringer Mannheim, Mannheim, Germany) and 50 IU of reverse transcriptase (Boehringer Mannheim). After incubation at 42°C for 1 h, one-fifth (4 µl) of the mixture subjected to RT was used as the template for PCR. PCR amplification was then carried out using the primers for human iNOS which spanned exons 22–26 (Geller et al., 1993); 5'-TGTCTGCAGCACATGGCTCAACAG-3' (3032–3055) and 5'-CTCAGATAATGCAGAGCTGGCTCC-3' (3737–3714) in a 20 µl PCR solution containing 10 mmol/l Tris–HCl (pH 8.3), 50 mmol/l KCl, 2 mmol/l MgCl2, 0.2 mmol/l of each dNTP, 0.5 µmol/l of each primer described above and 0.5 IU of Ex Taq polymerase (Takara) for 30 cycles (each cycle consisting of 94°C for 30 s, 55°C for 60 s and 72°C for 60 s) in a Perkin-Elmer GeneAmp 2400 Thermal Cycler (Foster City, CA, USA). In an attempt to increase the sensitivity of the detection of iNOS transcript, the cDNA obtained was assayed by nested PCR. Secondary PCR amplification was undertaken using the inner primers which spanned exons 23–26 (Geller et al., 1993); 5'-ATCCCTCCCATCCTTGCATCCTCAT-3' (3121–3145) and 5'-CTGTCCTTCTTCGCCTCGTAAGGAA-3' (3625–3601), and one-tenth (2 µl) of the primary amplified cDNA as the template. The cycle profile for the reaction consisted of 30 s at 94°C, 60 s at 65°C and 60 s at 72°C for 30 cycles. The amplified product was analysed by 1.5% agarose gel electrophoresis and ethidium bromide staining. The DNAs were transferred onto a Biodyne A membrane (Pall) for Southern blot analysis. The filter was hybridized with an [-32P] dCTP-labelled cDNA probe, and washed at high stringency. Autoradiography was carried out to confirm the identity of the PCR product. The PCR product was initially subcloned, ligated into pGEM-T vector (Promega) and subsequently sequence-verified.
Immunohistochemistry
Frozen sections (8 µm thick) were mounted onto silane-coated slides. The sections were then fixed in acetone at 4°C for 10 min, and washed with Dulbecco's phosphate-buffered saline (D-PBS) (Takara). Thereafter, the slides were incubated in 3% hydrogen peroxide in methanol to block endogenous peroxidase activity. The sections were preincubated with 2% normal goat serum in D-PBS at room temperature for 10 min. They were then incubated at room temperature for 1 h with an affinity column-purified anti-human iNOS polyclonal antibody raised against amino acids 1135–1153 (Santa Cruz, Heidelberg, Germany) at 1/100 dilution. Antibody binding was detected using a HistoStain-SP kit according to the manufacturer's instructions (Zymed, San Francisco, CA, USA). 3-amino-9-ethylcarbazol, which forms a reddish brown colour, was utilized as a chromogen. The sections were finally counterstained with Harris' haematoxylin and mounted in an aqueous medium.
Negative controls were applied for all tissue sections by replacement of the primary antibody with appropriately diluted isotype immunoglobulin G. Human DLD-1 colon adenocarcinoma cells (American Type Culture Collection, Rockville, MD, USA), which have previously been shown to express iNOS (Sherman et al., 1993), were stimulated for 9 h with 10 ng/ml of interleukin (IL)-1ß (Otsuka, Tokushima, Japan) and 500 IU/ml of interferon (IFN)-
(Chemicon, Temecula, CA, USA), and were used as the positive control.
Results
Detection of iNOS mRNA by Northern blot analysis or nested RT–PCR
Northern blot analysis of total RNA and poly(A)+ mRNA revealed a single 4.5 kbp band corresponding to the mRNA coding for human iNOS in all early pregnancy decidual and chorionic villous tissues examined (Figure 1). There was inter-individual variation in the intensities of iNOS mRNA signals in decidual and chorionic villous tissues, which was not related to the duration of gestation. On the other hand, iNOS transcripts could not be detected in the endometrium at any stage of the menstrual cycle by Northern blot analysis even after prolonged exposure (Figure 2). Amplification of RNA prepared from all endometrial tissues obtained at various stages of the menstrual cycle gave rise to a 505 bp PCR product corresponding to human iNOS (Figure 3). The bands representing amplification of RNA prepared from different tissues were of similar intensities.
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Immunohistochemical localization of iNOS
Immunolocalization of iNOS was confined to endometrial epithelial cells in the secretory endometrium (Figure 4
). Immunostaining for iNOS was negative in stromal cells in the endometrium. In the first trimester decidua, however, iNOS protein was localized in stromal cells as well as in glandular epithelial cells (Figure 5). Furthermore, strong immunostaining was observed in both syncytiotrophoblastic and cytotrophoblastic cells (Figure 6). There was no significant difference in staining localization among individual specimens, but there was inter-individual variation in staining intensities. No reaction product was present in control sections with substitution of the primary antibody by appropriately diluted isotype antibody (not shown). Specificity of iNOS antibody was confirmed in Western blot analysis, by which a 130 kDa band corresponding to the expected size for human iNOS antigen was detected (Yoshiki et al., 1999).
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Discussion
This study demonstrates that: (i) iNOS mRNA is detectable by Northern blot analysis in the first trimester decidual and chorionic villous tissues; (ii) iNOS mRNA is detectable in human endometrium throughout the menstrual cycle by nested RT–PCR but not by Northern blot analysis; (iii) iNOS protein is localized in epithelial cells; (iv) iNOS protein is not present in stromal cells, but comes to be expressed upon decidualization; and (v) iNOS protein is found in trophoblastic cells in the first trimester.
The question addressed by the present study concerns the expression of iNOS mRNA in human endometrium at various stages of the menstrual cycle and during early pregnancy. Previous studies using RT–PCR have reported the detection of iNOS mRNA in glandular epithelial cells (Telfer et al., 1997), in stromal cells (Yoshiki et al., 1999) and in the secretory phase endometrium (Tschugguel et al., 1998). However, there has been no study on the expression of iNOS mRNA in human endometrium during early pregnancy. The data we present here are the first demonstrating by Northern blot analysis that iNOS mRNA is expressed in human decidua and chorionic villi in the first trimester but not in the endometrium at any stage of the menstrual cycle. Our finding that endometrial iNOS mRNA concentration was increased in early pregnancy compared with that at different stages of the menstrual cycle supports the results obtained by Telfer et al. (1997), who demonstrated immunocytochemically localization of iNOS protein in decidualized stromal cells, but not in endometrial stromal cells during the menstrual cycle. Based on the report that the decidua in early pregnancy expresses a high concentration of cytokines (Saito et al., 1993), it may be assumed that the mechanism by which decidualization in vivo causes stromal cells to express high levels of iNOS mRNA is associated with exposure of cells to various cytokines. In fact, we have previously demonstrated markedly high levels of iNOS mRNA expression in human endometrial stromal cells cultured with combinations of IL-1ß and IFN- (Yoshiki et al., 1999). In contrast to the strong iNOS expression in the decidua and chorionic villi, the weak iNOS expression in the endometrium during the menstrual cycle may reflect the lack of sufficient exposure to cytokines.
There have been some studies on localization of iNOS in human placenta at term and during mid-trimester. However, there has been no study on iNOS expression in human chorionic villi in the first trimester. In human placenta at term, iNOS protein has been localized in the syncytiotrophoblast and the extravillous trophoblast, but not in the villous trophoblast, as determined by immunocytochemistry (Thomson et al., 1997). On the other hand, in human placenta during mid-trimester, iNOS mRNA has been present predominantly in the syncytiotrophoblast but also in the cytotrophoblast, as determined by in-situ hybridization (Baylis et al., 1999). We are the first to demonstrate that iNOS is strongly expressed in both syncytiotrophoblastic and cytotrophoblastic cells during early pregnancy. These studies show that iNOS expression in the cytotrophoblast decreases progressively during gestation. The presence of a large amount of iNOS at the feto–maternal interface throughout the first trimester of pregnancy suggests that iNOS may be an important factor involved in placentation and development of the embryo in early pregnancy.
One report has shown that rat pregnant uterus served as a source of NO, with iNOS as the major isoform involved in NO production (Dong et al., 1996), and that in early pregnancy the iNOS amount was considerably increased due to the presence of rat placental iNOS (Purcell et al., 1997), and also that iNOS mRNA was expressed in pregnant, but not in non-pregnant, uterus of the rat (Riemer et al., 1997). Additionally, Baylis et al. (1999) have reported that iNOS mRNA was constitutively expressed in mouse placenta. These findings show that NO produced by iNOS possibly has a close relation to uterine contractility during pregnancy. On the other hand, the expression of iNOS may have the advantage of permitting the integration of NO production into the complex processes required for the initiation of successful implantation and placentation (Norman and Cameron, 1996). Inducible NOS can catalyse the production of large quantities of NO (on the order of nanomoles), while the other two constitutive isoforms catalyse that of only small amounts (on the order of picomoles) (Berdeaux, 1993). The significance of the changes in endometrial iNOS expression can only be a subject of speculation at this stage. It is possible that NO produced in large quantities in the presence of iNOS plays a paracrine role in regulating uterine blood flow and immunosuppression for successful pregnancy.
In conclusion, the present study has been the first to demonstrate by Northern blot analysis that human endometrium in the first trimester of pregnancy constitutively expresses iNOS mRNA. The changes in iNOS expression we have described here are probably influenced by various cytokines present in human endometrium during early pregnancy. Further study will be required to determine whether NO plays a fundamental role in maintaining the biological environment of early pregnancy.
Acknowledgments
This study was supported by a Science Research Grant (11671599) to T.Kubota from the Ministry of Education, Science and Culture of Japan.
Notes
1 To whom correspondence should be addressed 
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Submitted on October 11, 1999; accepted on December 21, 1999.
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