Molecular Human Reproduction vol.5 no.4 pp. 353–357, 1999
Expression of inducible nitric oxide synthase in human cultured
endometrial stromal cells
Naoyuki Yoshiki1, Toshiro Kubota, Yukie Matsumoto and Takeshi Aso
Department of Obstetrics and Gynaecology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima,
Bunkyo-ku, Tokyo 113-8519, Japan
1To
whom correspondence should be addressed
The aim of the present study was to investigate the expression of mRNA and protein for inducible nitric
oxide synthase (iNOS) in human cultured endometrial stromal cells. The endometrial stromal cells were
cultured in the absence or the presence of cytokines such as interleukin (IL)-1β and interferon (IFN)-γ, which
are inherently detectable in the endometrium. Nested reverse transcription–polymerase chain reaction
detected iNOS mRNA in stromal cells cultured without cytokines. Northern blot analysis failed to detect
iNOS mRNA in stromal cells cultured for 9 h in the absence of cytokines or in the presence of IL-1β (10–100
ng/ml) or IFN-γ (10–1000 U/ml) alone, but could detect iNOS mRNA cultured in combinations of IL-1β and
IFN-γ. The concentrations of iNOS mRNA were increased as early as 3 h after the addition of the cytokine
combination and persisted for up to 36 h. Western blot analysis demonstrated iNOS protein in stromal cells
cultured for 12 h with combined IL-1β and IFN-γ. These results raise the possibility that nitric oxide locally
synthesized by iNOS may be involved in the control of endometrial functions.
Key words: endometrial stromal cells/inducible nitric oxide synthase/interferon-γ/interleukin-1β
Introduction
Nitric oxide (NO), one of the smallest and simplest biosynthetic
products, is a free radical gas which acts especially within the
vascular system (Palmer et al., 1987). It is also 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 signalling molecule is therefore likely to have an
important role within the endometrium which shows changes
in vascular function throughout the course of the menstrual
cycle and at the time of implantation.
NO is made by conversion of L-arginine to L-citrulline
catalysed by a family of enzymes known as nitric oxide
synthases (NOSs) (Moncada et al., 1991). NOS isoforms are
categorized into three types, i.e. neuronal (n) NOS, endothelial
(e) NOS and inducible (i) NOS. nNOS, originally identified
in neuronal tissues, is expressed constitutively and is calciumdependent, similar to eNOS, which was first identified as a
constitutive enzyme in vascular endothelial cells. On the other
hand, iNOS, which is not dependent on calcium concentrations,
has been shown to be expressed in a wide array of tissues and
organs, and can be induced by cytokines such as interleukin
(IL)-1β, interferon (IFN)-γ and tumour necrosis factor (TNF)α or endotoxins such as bacterial lipopolysaccharide (LPS)
(Förstermann et al., 1995). Inducible NOS is normally absent
from cells under resting conditions, but when stimulated by
cytokines or LPS, it can produce large quantities of NO
(nanomoles), while the other two constitutive isoforms produce
small amounts of NO (picomoles) for short periods
(Berdeaux, 1993).
© European Society of Human Reproduction and Embryology
The production of NO in the endometrium has not been
widely addressed. Using immunohistochemistry, nNOS protein
has been detected in endometrial epithelium in the rat (Schmidt
et al., 1992) and iNOS in the mouse (Huang et al., 1995).
There has been a previous study in humans that showed
protein and mRNA for eNOS detected in endometrial glandular
epithelium and stroma by in-situ hybridization and immunocytochemistry (Telfer et al., 1995). Moreover, eNOS mRNA
has been shown in human epithelial glands throughout the
menstrual cycle by Northern blot analysis, whilst the expression
of iNOS mRNA has been detected only in the epithelial
glands of a menstrual endometrium by solution hybridization/
ribonuclease protection assay (Tseng et al., 1996). In addition,
recent work has reported that protein and mRNA for eNOS
and iNOS were detected in human endometrial glandular
epithelium but not found in endometrial stroma by immunocytochemistry and reverse transcription–polymerase chain
reaction (RT–PCR) (Telfer et al., 1997). Thus the role of NOS
in the control of endometrial functions has not been fully
understood.
The aim of the present study was to detect the presence of
iNOS mRNA in human cultured endometrial stromal cells by
RT–PCR. We further examined whether mRNA and protein
for iNOS could be induced in cultured stromal cells by
two kinds of cytokines, which are inherently present in the
endometrium, using Northern blot and Western blot analysis.
Materials and methods
Chemicals
Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine
serum (FBS), bovine serum albumin (BSA), collagenase, deoxyribonu-
353
N.Yoshiki et al.
clease, antibiotic antimycotic solution and Dulbecco’s phosphatebuffered saline (D-PBS) were purchased from Gibco (Grand Island,
NY, USA). Recombinant human IL-1β was a generous gift from
Otsuka Pharmaceutical Co. (Tokushima, Japan) and recombinant
human IFN-γ was obtained from Chemicon Internatinal Inc.
(Temecula, CA, USA), respectively. All other chemicals were of
analytical grade from commercial sources.
Tissue collection
Human endometrial tissues in the secretory phase were obtained from
premenopausal women with regular menstrual cycles undergoing
hysterectomy for benign diseases at the Tokyo Medical and Dental
University Hospital, Japan. The stage of the cycle was determined
by the date of the last menstrual period. With the informed consent
of each woman, all of the sampling and using of these tissues for
this study were approved by the local ethics committee. All the
patients had no prior hormonal drug treatment and were otherwise
healthy. Endometrial tissue was scraped from the uterine wall with
scissors and pincette immediately after hysterectomy, thoroughly
washed with physiological saline, placed in cold RPMI 1640 medium
without FBS, and rapidly transported to the laboratory.
Isolation of endometrial stromal cells and cell culture
The specimen was washed three times with RPMI 1640 medium to
eliminate blood clots and then cut into multiple pieces. According to
the methods described previously (Satyaswaroop et al., 1979), tissue
pieces were treated with 2 mg/ml collagenase and 50 µg/ml deoxyribonuclease in D-PBS and digested at 37°C for 1 h. The digested
endometrial cells were filtrated through 38 µm steel mesh to remove
cell debris and epithelial gland fragments, and washed with D-PBS
supplemented with 2 mM EDTA and 0.5% BSA three times. To remove
macrophages/monocytes, the cells were treated with MicroBeads
conjugated with monoclonal anti-human CD14 antibodies (Miltenyi
Biotec, Bergisch Gladbach, Germany), followed by Magnetic Cell
Sorting depletion column chromatography according to the manufacturer’s instructions (Miltenyi Biotec). The number of viable endometrial cells was counted by Trypan Blue exclusion. Viable cells
were seeded onto 100 mm plastic culture dishes (Falcon, Rockford,
IL, USA) at a density of 53106 cells/dish in 5 ml of RPMI 1640
medium containing 10% FBS and antibiotics. The medium was
removed 2 h later, and adherent cells were washed with fresh medium.
Quick attachment of stromal cells selectively removed blood cell
contamination, and cells were cultured in a humidified atmosphere
of 5% CO2 in 95% air at 37°C. Cultured cells were used for
experiments when 70% confluence was achieved. On the day of the
experiment, microscopic inspection revealed that almost all cultured
cells were stromal/fibroblast-like cells in appearance without
epithelial contamination. This result was immunocytochemically
confirmed by positive staining with anti-vimentin antibody and not
with anti-cytokeratin antibody. The medium was removed, endometrial stromal cells were washed and treated with various concentrations of the following cytokines; IL-1β (10–100 ng/ml) and IFN-γ
(10–1000 U/ml). They were maintained for various periods up to 36 h.
RNA extraction and nested RT–PCR
Total RNA was isolated from cultured cells by the single step
acid guanidinium thiocyanate–phenol–chloroform extraction method
(Chomczynski and Sacchi, 1987). Complementary DNA synthesis
was carried out on 1 µg of total RNA as follows: RNA dissolved in
sterile DEPC-treated water was heated to 80°C with 50 ng of random
hexanucleotide primers (Takara, Otsu, Japan) for 5 min, then added
to a total volume of 20 µl of reaction mixture containing 50 mM
Tris–HCl (pH 8.3), 40 mM KCl, 5 mM MgCl2, 0.5% Tween-20
354
(v/v), 10 mM DTT, 1 mM each dNTP, 20 units of ribonuclease
inhibitor (Boehringer Mannheim, Mannheim, Germany) and 50 units
of reverse transcriptase (Boehringer Mannheim). After incubation at
42°C for 1 h, one-fifth (4 µl) of the RT reacted mixture was used as
template for PCR. PCR amplification was then carried out with the
primers for human iNOS which spanned exons 22–26 (Geller et al.,
1993); 59-TGTCTGCAGCACATGGCTCAACAG-39 (3032–3055)
and 59-CTCAGATAATGCAGAGCTGGCTCC-39 (3737–3714) in a
total volume of 20 µl of PCR solution containing 10 mM Tris–HCl
(pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM each dNTP, 0.5 µM
each primer described above and 0.5 units of Ex Taq polymerase
(Takara) for 30 cycles (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 for the detection
of iNOS transcript, cDNA obtained was assayed by a nested PCR
method. For this reason, second PCR amplification was undertaken
with the inner primers which spanned exons 23–26 (Geller et al.,
1993); 59-ATCCCTCCCATCCTTGCATCCTCAT-39 (3121–3145)
and 59-CTGTCCTTCTTCGCCTCGTAAGGAA-39 (3625–3601), and
one-tenth (2 µl) of the first amplified cDNA as template. The reaction
was carried out in a cycle profile of 30 s at 94°C, 60 s at 65°C and
60 s at 72°C for 30 cycles. Amplified product was analysed by 1.5%
agarose gel electrophoresis and ethidium bromide staining. The PCR
product was gel-purified using a QIAEX II kit (Qiagen, Hilden,
Germany) and isolated cDNA was cloned into pGEM-T vector
according to the instructions provided by the manufacturer (Promega,
Madison, WI, USA). The sequence of the cloned cDNA was analysed
with an automated ABI PRISM 310 sequencer (Perkin–Elmer).
Northern blot analysis
Cloned cDNA fragment containing 505 bp of human iNOS-specific
sequence was used as a probe for Northern blot analysis. Twenty µg
of total cellular RNA was denatured and subjected to electrophoresis
in 1% agarose/5.4% formaldehyde gel in 13MOPS buffer (20 mM
MOPS, 50 µM sodium acetate, 1 mM 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 a 25 ng 32P-labelled probe in QuikHyb Hybridization
Solution (Stratagene, La Jolla, CA, USA) at 68°C for 1 h. The
membrane was washed initially with 23standard saline citrate (SSC;
13SSC 5 150 mM NaCl, 15 mM sodium citrate)/0.1% sodium
dodecyl sulphate (SDS) at room temperature for 15 min twice,
followed by increasing stringency up to 0.13SSC/0.1% SDS at 60°C
for 30 min and exposed to an imaging plate (Fuji Photo Film Co.,
Japan). The relative intensities of Northern blot hybridization signals
were determined using a Bio-imaging analyser BAS2000 (Fuji Photo
Film Co.).
Western blot analysis
The cultured stromal cells were fixed with 10% trichloroacetic acid
in D-PBS and lysed with 20 µl of lysis buffer (9 M urea/2% Triton
X-100/1% DTT), then supplemented with 5 µl of 10% LiDS. The
concentration of protein in lysates was measured with a BCA protein
assay kit (Pierce, Rockford, IL, USA). Each sample containing
equivalent amount of protein was separated on 7.5% SDS–polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene
difluoride membrane (Atto, Tokyo, Japan). After blocking with Block
Ace (Dainippon, Osaka, Japan), the membrane was incubated with
an affinity column-purified anti-human iNOS polyclonal antibody
(1:1000) raised against amino acids 1135–1153 (Santa Cruz, Heidelberg, Germany), followed by incubation with a horseradish peroxidase
conjugated anti-rabbit IgG antibody (1:3000) (Amersham, Buckinghamshire, UK). The bound antibody was detected by the enhanced
Expression of iNOS in human endometrial stromal cells
Figure 1. Nested reverse transcription–polymerase chain reaction
for iNOS (inducible nitric oxide synthase) using total RNA
extracted from human endometrial stromal cells cultured without
cytokines. A 505 bp polymerase chain reaction product was the
size predicted based on the cDNA sequence of human iNOS. The
amplified cDNA fragment was separated on a 1.5% agarose gel and
visualized by ethidium bromide staining.
chemiluminescence detection system according to the recommended
procedure (Amersham).
Figure 2. Effect of interleukin (IL)-1β and/or interferon (IFN)-γ on
iNOS (inducible nitric oxide synthase) mRNA expression in human
cultured endometrial stromal cells with different treatments of
cytokines. iNOS mRNA was induced by combinations of IL-1β and
IFN-γ, and its peak was at a concentration of 10 ng/ml of IL-1β
and 500 U/ml of IFN-γ. The positive control for human iNOS
mRNA is indicated in the right-hand lane; total RNA isolated from
DLD-1 colon adenocarcinoma cells stimulated for 9 h with IL-1β
(10 ng/ml) and IFN-γ (500 U/ml). The positions of the 18S and
28S ribosomal RNA submits are indicated at the right.
Results
Detection of iNOS mRNA in endometrial stromal cells
cultured without cytokines by nested RT–PCR
Messenger RNA for iNOS was detected by nested RT–PCR
in total RNA extracted from human endometrial stromal cells
cultured without cytokines. As shown in Figure 1, nested RT–
PCR for iNOS in cultured cells produced one intense band of
the expected size and sequencing of the amplified product
revealed 99% homology with the published sequence of human
iNOS (Geller et al., 1993).
Effect of cytokines on iNOS mRNA expression in
cultured endometrial stromal cells
To examine whether iNOS mRNA was induced by cytokines
in cultured endometrial stromal cells, total RNA extracted
from cells was analysed by Northern blot hybridization. As
shown in Figure 2, iNOS mRNA was not detected in stromal
cells cultured for 9 h in either the absence of cytokines or the
presence of IL-1β or IFN-γ alone. However, combinations of
IL-1β and IFN-γ increased the amount of iNOS mRNA in a
dose-dependent manner on IFN-γ, and the expression
reached its peak at a concentration of 10 ng/ml of IL-1β and
500 U/ml of IFN-γ. A prominent transcript of iNOS mRNA
was ~4.5 kilobases in length.
Figure 3 shows the time-course of 10 ng/ml IL-1β and
500 U/ml IFN-γ stimulation for 0, 3, 6, 9, 12, 18, 24 and 36
h. The expression of iNOS mRNA began as early as 3 h after
the addition of the cytokine combination and continued for up
to 36 h.
Detection of iNOS protein by Western blot analysis
As shown in Figure 4, iNOS protein was produced in stromal
cells cultured for 12 h in the presence of 10 ng/ml of IL-1β
and 500 U/ml of IFN-γ, but was undetectable when cells were
incubated without cytokines. A 130 kD protein band was
detected corresponding to the appropriate molecular weight
Figure 3. Northern blot analysis of the time-course of iNOS
(inducible nitric oxide synthase) mRNA expression in human
cultured endometrial stromal cells stimulated with combined
interleukin (IL)-1β (10 ng/ml) and interferon (IFN)-γ (500 U/ml).
RNA isolated from DLD-1 colon adenocarcinoma cells stimulated
for 9 h with IL-1β (10 ng/ml) and IFN-γ (500 U/ml) served as
positive control. The positions of the 18S and 28S ribosomal RNA
submits are indicated at the right.
for human iNOS protein. A smaller non-specific band was
also detected in both samples.
Discussion
This study is the first to demonstrate the detection of iNOS
mRNA by nested RT–PCR in human endometrial stromal cells
cultured without cytokines. Although RT–PCR (not nested
RT–PCR) detected iNOS mRNA in our preliminary studies, it
produced a few non-specific bands (not shown). A previous
study has already reported the detection of iNOS mRNA by
RT–PCR in the epithelial cells of human endometrial gland
fragments (Telfer et al., 1997). The clear amplification obtained
in epithelial cells but not in stromal cells by RT–PCR might
be due to the levels of iNOS mRNA in each component of
the endometrium or the different PCR conditions. From the
355
N.Yoshiki et al.
Figure 4. Western blot analysis of iNOS (inducible nitric oxide
synthase) protein using an anti-human iNOS polyclonal antibody
against proteins obtained from human cultured endometrial stromal
cell lysates. The antibody reacted with a protein band at 130 kD
(appropriate molecular weight for human iNOS protein), when
stromal cells were cultured for 12 h with interleukin (IL)-1β
(10 ng/ml) and interferon (IFN)-γ (500 U/ml).
previous (Telfer et al., 1997) and the present study, it is now
concluded that endometrial iNOS mRNA is detectable in both
epithelial and stromal cells.
Using Northern blot analysis, we have also been the first to
demonstrate that a considerable amount of iNOS mRNA is
expressed in stromal cells cultured in combinations of IL-1β
and IFN-γ. However, no iNOS mRNA expression could be
detected in either the absence of cytokines or the presence of
either IL-1β or IFN-γ alone. It has been well established
that iNOS is regulated mainly at the transcriptional level
(Förstermann et al., 1995). The structural similarity among
the known human and murine promoter fragments contrasts
with the different cytokine patterns required to induce human
or murine iNOS (Nunokawa et al., 1994). Therefore, the
cytokines or the combinations of cytokines that produce
significant iNOS expression vary between species or between
cell types within the same species. For example, in rat
reproductive organs, one report has shown that IL-1β alone
stimulated iNOS mRNA expression in cultured Leydig cells
(Tatsumi et al., 1997). On the other hand, another study has
demonstrated that Northern blot analysis failed to detect iNOS
mRNA in granulosa cells cultured with a single cytokine, such
as IL-1β, IFN-γ and TNF-α, but that the combinations of any
two or three cytokines increased the amount of iNOS mRNA
(Matsumi et al., 1998). The present study has revealed that
the combined administration of IL-1β and IFN-γ induces
mRNA and protein for iNOS in human cultured endometrial
stromal cells, with the expression beginning as early as 3 h
and persisting for up to 36 h. Thus we conclude that iNOS in
human endometrial stromal cells has few physiological functions in vivo but endometrial stromal cells have the ability to
express mRNA and protein for iNOS in the presence of
cytokines, which are inherently detectable in the endometrium
especially in the secretory phase.
It has been previously reported that progesterone increased
uterine NO formation during pregnancy in the rat (Yallampalli
et al., 1994), and recent work has revealed that antiprogestins
356
reduced iNOS protein in rat uterus (Dong et al., 1996). There
has been a previous study showing that immunoreactive iNOS
was no longer detectable in the epithelial cells in the uterus
of ovariectomized mice following treatment with 17β-oestradiol, but that weak staining of iNOS was observed in the
epithelium as a consequence of progesterone treatment (Huang
et al., 1995). These findings suggest that iNOS activity in vivo
is closely related to hormonal regulation (mainly progesterone)
as well as cytokine stimulation. Furthermore, one report has
shown that the pregnant rat uterus served as a source of
NO that acted as an autocrine mediator in maintaining the
quiescence necessary for normal fetal development, with iNOS
appearing to be the major isoform involved in NO production
(Dong et al., 1996), and another has demonstrated that early
in the pregnancy the iNOS amount was considerably increased
by the presence of rat placental iNOS and that its expression
was down-regulated prior to term, to reach very low values
during delivery (Purcell et al., 1997). However, there has been
a study in human uterus and placenta that showed no difference
in either the expression or enzyme activity of iNOS before
and during labour at term, because iNOS may be already
down-regulated near term (Thomson et al., 1997).
We have demonstrated that a certain combination of cytokines induced mRNA and protein for iNOS in human cultured
endometrial stromal cells. Telfer et al. (1997) have shown
that immunoreactivity for iNOS was not present in human
endometrial stroma throughout the menstrual cycle, but that
iNOS-like immunoreactivity was seen in decidualized stromal
cells following treatment with exogenous progestogen and in
tissues obtained in the first trimester of pregnancy. The
mechanisms by which decidualization in vivo causes stromal
cells to express iNOS protein (Telfer et al., 1997) appear to
be associated with exposure of cells to progesterone and
various cytokines, because decidua in the early pregnancy
expresses a high concentration of cytokines (Saito et al., 1993).
Our results indicate that stromal cells in the secretory phase
have the potential to express iNOS in the presence of cytokines
before decidualization. Therefore, because the proportion of
leukocytes in human endometrial stroma increases dramatically
in the late secretory phase and in the early pregnancy (Bulmer
et al., 1991), there is a possibility of iNOS expression in
stromal cells locally around blood vessels in the late secretory
phase under the stimulation by cytokines derived from infiltrating cells, such as macrophages and natural killer cells. Thus
the expression of iNOS may have the advantage of permitting
the integration of NO production into the complex networks
required for the initiation of successful implantation and
placentation (Norman and Cameron, 1996).
What is the effect of NO on successful pregnancy? Nitric
oxide is usually viewed as a very short-acting substance that
is synthesized within close proximity to the site of its activity.
Its half-life is only a few seconds. However, it is known that
NO can rapidly diffuse to distances of 200–400 µm within
tissues (Lancaster, 1994). This is well within the range for
stromal NO to act on the vascular bed. Therefore, NO produced
by iNOS may be involved in controlling the development of
the microvascular system and vascular tone in the endometrium
in the secretory phase and in the early pregnancy.
Expression of iNOS in human endometrial stromal cells
What is another physiological role of NO in endometrial
functioning? Lin et al. (1998) have demonstrated that the NO/
NOS system plays a role in the control of endothelin secretion
via the action of cytokines, and recent findings have revealed
that endothelin has potential autocrine and/or paracrine functions in human endometrial cells (Kubota et al., 1995). The
finding that NOS activity has a close relation to the control of
endothelin release in human endometrium suggests that the
NO/NOS system may play an important role in the local
regulation of endometrial functions.
In conclusion, the present study has demonstrated the
expression of mRNA and protein for iNOS in human endometrial stromal cells cultured with combined IL-1β and IFN-γ.
Further study will be required to examine how NO modulates
the endometrial milieu.
Acknowledgements
This study was supported by a Science Research Grant (08671873)
to T.Kubota from the Ministry of Education, Science and Culture
of Japan.
References
Berdeaux, A. (1993) Nitric oxide: an ubiquitous messenger. Fundam. Clin.
Pharmacol., 7, 401–411.
Bulmer, J.N., Morrison, L., Longfellow, M. et al. (1991) Granulated
lymphocytes in human endometrium: histochemical and immunohistochemical studies. Hum. Reprod., 6, 791–798.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal.
Biochem., 162, 156–159.
Dong, Y-L., Gangula, P.R.R. and Yallampalli, C. (1996) Nitric oxide synthase
isoforms in the rat uterus: differential regulation during pregnancy and
labour. J. Reprod. Fertil., 107, 249–254.
Förstermann, U., Gath, I., Schwarz, P. et al. (1995) Isoforms of nitric oxide
synthase: properties, cellular distribution and expressional control. Biochem.
Pharmacol., 50, 1321–1332.
Geller, D.A., Lowenstein, C.J., Shapiro, R.A. et al. (1993) Molecular cloning
and expression of inducible nitric oxide synthase from human hepatocytes.
Proc. Natl. Acad. Sci. USA, 90, 3491–3495.
Huang, J., Roby, K.F., Pace, J.L. et al. (1995) Cellular localization and
hormonal regulation of inducible nitric oxide synthase in cycling mouse
uterus. J. Leukocyte Biol., 57, 27–35.
Kubota, T., Taguchi, M., Kamada, S. et al. (1995) Endothelin synthesis and
receptors in human endometrium throughout the normal menstrual cycle.
Mol. Hum. Reprod., 1, see Hum. Reprod., 10, 2204–2208.
Lancaster, J.R. Jr (1994) Simulation of the diffusion and reaction of
endogenously produced nitric oxide. Proc. Natl. Acad. Sci. USA, 91,
8137–8141.
Lin, Z., Kubota, T., Masuda, M. et al. (1998) Role of nitric oxide synthase
in release of endothelin from cultured human endometrial cells. Eur. J.
Endocrinol., 138, 467–471.
Matsumi, H., Yano, T., Koji, T. et al. (1998) Expression and localization of
inducible nitric oxide synthase in the rat ovary: a possible involvement of
nitric oxide in the follicular development. Biochem. Biophys. Res. Commun.,
243, 67–72.
Moncada, S., Palmer, R.M.J. and Higgis, E.A. (1991) Nitric oxide: physiology,
pathophysiology, and pharmacology. Pharmacol. Rev., 43, 109–142.
Nathan, C. (1992) Nitric oxide as a secretory product of mammalian cells.
FASEB J., 6, 3051–3064.
Norman, J.E. and Cameron, I.T. (1996) Nitric oxide in the human uterus. Rev.
Reprod., 1, 61–68.
Nunokawa, Y., Ishida, N. and Tanaka, S. (1994) Promoter analysis of
human inducible nitric oxide synthase gene associated with cardiovascular
homeostasis. Biochem. Biophys. Res. Commun., 200, 802–807.
Palmer, R.M.J., Ferrige, A.G. and Moncada, S. (1987) Nitric oxide release
accounts for the biological activity of endothelium-derived relaxing factor.
Nature, 327, 524–526.
Purcell, T.L., Buhimschi, I.A., Given, R. et al. (1997) Inducible nitric oxide
synthase is present in the rat placenta at the fetal–maternal interface and
decreases prior to labour. Mol. Hum. Reprod., 3, 485–491.
Saito, S., Nishikawa, K., Morii, T. et al. (1993) Cytokine production by
CD16–CD56bright natural killer cells in the human early pregnancy decidua.
Int. Immunol., 5, 559–563.
Satyaswaroop, P.G., Bressler, R.S., de la Pena, M.M. et al. (1979) Isolation
and culture of human endometrial glands. J. Clin. Endocrinol. Metab., 48,
639–641.
Schmidt, H.H.H.W., Gagne, G.D., Nakane, M. et al. (1992) Mapping of neural
nitric oxide synthase in the rat suggests frequent co-localization with
NADPH diaphorase but not with soluble guanylyl cyclase, and novel
paraneural functions for nitrinergic signal transduction. J. Histochem.
Cytochem., 40, 1439–1456.
Tatsumi, N., Fujisawa, M., Kanzaki, M. et al. (1997) Nitric oxide production
by cultured rat Leydig cells. Endocrinology, 138, 994–998.
Telfer, J.F., Lyall, F., Norman, J.E. et al. (1995) Identification of nitric oxide
synthase in human uterus. Hum. Reprod., 10, 19–23.
Telfer, J.F., Irvine G.A., Kohnen, G. et al. (1997) Expression of endothelial
and inducible nitric oxide synthase in non-pregnant and decidualized human
endometrium. Mol. Hum. Reprod., 3, 69–75.
Thomson, A.J., Telfer, J.F., Kohnen, G. et al. (1997) Nitic oxide synthase
activity and localization do not change in uterus and placenta during human
parturition. Hum. Reprod., 12, 2546–2552.
Tseng, L., Zhang, J., Peresleni, T.Y. et al. (1996) Cyclic expression of
endothelial nitric oxide synthase mRNA in the epithelial glands of human
endometrium. J. Soc. Gynecol. Invest., 3, 33–38.
Yallampalli, C., Byam-Smith, M., Nelson, S.O. et al. (1994) Steroid hormones
modulate the production of nitric oxide and cGMP in the rat uterus.
Endocrinology, 134, 1971–1974.
Received on August 18, 1998; accepted on December 18, 1998
357