The role of nitric oxide in the effects of ovarian steroids on

spontaneous myometrial contractility in rats

A. Bulbul a,*, A. Yagcı b, K. Altunbas b, A. Sevimli c, H.A. Celik d,A. Karadeniz e, E. Akdag f

a Department of Physiology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyon, Turkeyb Department of Histology & Embryology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyon, Turkey

c Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyon, Turkeyd Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyon, Turkey

e Department of Physiology, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkeyf Department of Laboratory Animals, Gulhane Military Medical Academy Research Center, Ankara, Turkey

Received 12 December 2006; received in revised form 26 July 2007; accepted 2 August 2007

www.theriojournal.com

Theriogenology 68 (2007) 1156–1168

Abstract

Forty ovariectomized rats were apportioned into one control and three experimental groups (n = 10 each) to evaluate the role

of nitric oxide in the effects of ovarian steroids on spontaneous myometrial contractility in rats. The control group (group Ov)

received sesame oil once daily for 10 days, whereas rats in the experimental groups were treated with progesterone (2 mg/

(rat day); group P), 17b-estradiol (10 mg/(rat day); group E2), or progesterone and 17b-estradiol together (group E2 + P). The

functionality of the arginine–nitric oxide synthase (NOS)–nitric oxide (NO) pathway in the uterine horns of sacrificed rats was

evaluated in an isolated organ bath. L-Arginine, sodium nitroprusside (SNP) and 8-Br-cGMP decreased uterine contractile

tension induced by electric field stimulation (EFS) in the Ov, P, and E2 + P groups, but not in the E2 group. In addition, L-

arginine was ineffective when applied together with a NOS inhibitor, L-nitro-N-arginine (L-NNA). The percentage of contractile

inhibition was higher in the Ov and P groups compared to the E2 + P group. Immunohistochemical evaluation revealed that

expression of neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) in smooth muscles and nerve cells

did not differ among the groups. Expression of nNOS and eNOS was strongly evident in the E2 and E2 + P groups at both

surface and glandular epithelium of the endometrium. iNOS expression was increased in surface epithelium of the E2 and

E2 + P groups. However, iNOS expression was only increased in glandular epithelial cells of the E2 + P group. In conclusion,

the L-arginine–NOS–NO pathway inhibits myometrial contractions via cGMP-dependent and -independent mechanisms, and

while progesterone maintains the nitric oxide effects, estrogen prevents them. These results suggest that NOS does not mediate

the effects of estrogen.

# 2007 Elsevier Inc. All rights reserved.

Keywords: Nitric oxide; Myometrial contractility; Organ bath; Ovarian steroids; Nitric oxide synthase

* Corresponding author. Tel.: +90 2722281312;

fax: +90 2722281349.

E-mail address: abulbul@aku.edu.tr (A. Bulbul).

0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.theriogenology.2007.08.014

1. Introduction

Ovarian steroids are one of the most important

factors affecting uterine morphology and motility. The

characteristics of spontaneous myometrial contractility

change due to direct actions of these hormones. Uterine

A. Bulbul et al. / Theriogenology 68 (2007) 1156–1168 1157

function and activity changes depending on the stage of

the estrus cycle or stage of pregnancy [1]. During the

follicular period, when estrogen levels are high, the

amplitude of uterine contraction increases in humans

[2,3]. Moreover, increased contractile tension following

17b-estradiol treatment has been demonstrated in vitro

in ovariectomized rats [4]. On the other hand,

progesterone has been reported to downregulate

prostaglandins, oxytocin receptors, and calcium chan-

nels, thus decreasing the occurrence and tension of

contraction [5,6].

In recent years, nitric oxide has been reported to

mediate the effects of sex hormones on uterine motility

[7]. Endogenous nitric oxide is derived from the amino

acid L-arginine by NOS [8]. Similarly, an exogenous

nitric oxide effect can be derived from nitrate-

containing compounds such as sodium nitroprusside

(SNP) [9]. One function of nitric oxide in reproductive

physiology is to regulate spontaneous uterine con-

tractility [10]. The level of nitric oxide synthesized via

NOS varies with different physiologic and pathologic

circumstances, thereby causing the patterns of sponta-

neous uterine contractility to change [11–13]. It is

known from in vitro studies that nitric oxide decreases

[8] or inhibits [9] uterine contractility depending on the

stages of menstrual period or gestation. Nitric oxide

inhibits the contractility of smooth muscles via

activating guanylate cyclase (GC), which is diffused

into smooth muscle cells [10]. Activated GC increases

cGMP levels, and consequently relaxes smooth

muscles [14].

NOS has inducible (iNOS) and constitutive (cNOS,

which includes nNOS and eNOS) isoforms, which are

present at various levels in different uterine tissues

[11,13]. eNOS levels in uterine vessels of humans and

rats increase during pregnancy [15]. Similarly, eNOS

activity in uterine vessels is higher during the

follicular phase of the estrus cycle than the luteal

phase [16]. Because estrogen increases nitric oxide

levels in genital organs and in circulating blood, it has

been suggested that estrogen could be responsible for

the increase in nitric oxide levels during pregnancy

and the follicular phase of the estrus cycle [15]. It has

been reported that the distribution of NOS and

changes in its activity in the female reproductive

system may play a role in the regulation of smooth

muscle contractility [17]. Therefore, the aim of this

study was to evaluate in vitro the role of nitric oxide in

the effects of ovarian steroids on spontaneous

myometrial contractility, and determine the effects

of ovarian steroids on localization of NOS protein in

vivo in the rat uterus.

2. Materials and methods

2.1. Animals

Female, Sprague–Dawley rats were obtained from

Gulhane Military Medical Academy Research Center,

Department of Laboratory Animals. Rats in all groups

were fed ‘‘ad libitum’’ with the same commercial rat

diet. The ethics committee of Afyonkocatepe Uni-

versity approved all procedures.

The ovariectomy procedure was performed at 12

weeks of age (n = 40). Rats were anesthetized by an

intra-peritoneal ketamine (21.2 mg/kg) and xylazine

(4.2 mg/kg) combination. Small bilateral incisions

were made on the dorsum to expose the ovaries

retroperitoneally. The ovarian vessels were then

clamped and the ovaries removed. Afterwards, the

uterine tubes were ligated and the muscles and skin

were sutured.

Two weeks after operation, the ovariectomized rats

were randomly assigned to four groups of 10 rats each.

Rats in the Ov group received sesame oil once daily for

10 days, whereas rats in the experimental groups were

treated with progesterone 2 mg/(rat day) (group P),

17b-estradiol 10 mg/(rat day) (group E2), and a

combination of progesterone and 17b-estradiol at the

same respective dosages (group E2 + P).

The hormone treatments were continued for 10 days,

after which the rats were killed by cervical dislocation.

Immediately after death, the abdominal cavities of the

rats were opened and the uterine horns were removed.

Each individual horn was placed in Krebs solution

(118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1 mM

MgSO4, 1 mM KH2PO4, 25 mM NaHCO3, and 11 mM

glucose). Subsequently, the surrounding mesentery and

fat tissues were carefully removed from the uterus, and

a strip-shaped tissue sample of the uterus with

dimensions of 0.1–0.3 cm � 1.0 cm was obtained.

One edge of each uterine tissue preparation was fixed

to platinum ring electrodes. The opposite edge of the

tissue was connected to a force–displacement transdu-

cer (model 10-A; MAY, Commat, Ankara, Turkey).

Isolated uterine tissues were placed in organ baths filled

with 20 mL Krebs solution, which were continuously

ventilated with gas mixture (95% O2–5% CO2) at 37 8C.

The isometric smooth muscle activity of the uterine

samples were monitored and recorded by computer via

the force transducer and an acquisition system (model

MP30 WSW with Biopac Student Lab PRO Software,

Biopac Systems). An EFS device (model ISO 150-C,

MAY, Commat, Ankara, Turkey) was used for electrical

stimulation.

A. Bulbul et al. / Theriogenology 68 (2007) 1156–11681158

Table 1

Experimental procedures applied to isolated uterine tissues in organ baths

Treatment Treatment procedure Purpose

Incubation Incubated in Krebs solution for 1 h Adaptation

Treatment I Electric stimulation (EFS) Determination of submaximal contraction

Treatment II L-Arginine (10�5 M, 10�4 M, 10�3 M) + EFS Determination of endogenous NO effect

L-NNA (10�3 M) + L-arginine (10�3 M) + EFS Determination of endogenous NO effect on enzymatic pathway

MB (10�5 M) + L-arginine (10�3 M) + EFS Inhibition of GC pathway

Treatment III SNP (10�8 M, 10�7 M, 10�6 M, 10�5 M) + EFS Determination of exogenous NO effect

Treatment IV 8-Br-cGMP (10�8 M, 10�7 M, 10�6 M) + EFS Determination of cGMP effect

2.2. Recording of isometric uterine contractility

Uterus samples in organ baths were kept in Krebs

solution for at least 1 h prior to the recordings to enable

the tissues to adapt to the environment, and the solution

was refreshed in 15-min intervals.Theappropriate resting

tension for the strips was determined in initial experi-

ments. Strips were placed under progressive increments

of tension, and contractile responses to KCl (90 mM in

Krebs solution) were measured under the various resting

tension conditions. Optimal tension relationships were

achieved with resting tensions of 1 g for the uterus strips.

Therefore, a resting tension of 1 g was applied to the

tissues. EFS was then used to elicit neurogenic responses

[18], carried out using single square wave pulses

(durations 0.25, 0.5 and 1 ms) at various frequencies

(2, 4, 8, 32 and 64 Hz). The voltage giving maximal

response was determined individually for each strip.

The experimental procedure applied to the tissue

samples is summarized in Table 1. In order to determine

endogenous nitric oxide activity, L-arginine solution

(1 mM to 1 mM) was added to the Krebs solution. SNP

(0.1 mM to 10 nM) was used for evaluation of the

exogenous nitric oxide pathway. 0.1 mM to 10 nM doses

of 8-Br-cGMP (an analogue for determining the effects of

cGMP) were added in logarithmically increasing

quantities every 10 min. L-Arginine (1 mM) was added

following 100 mM of L-NNA (nitro-N-arginine) treat-

ment in order to determine the effect of endogenous NO

on the enzymatic pathway. To evaluate the effect on the

guanyl cyclase pathway, L-arginine (1 mM) was applied

to the samples after incubation in methylene blue (MB)

for 30 min. All applications were performed on the same

uterus.

2.3. Determination of distribution of NOS in

uterine tissues

Immunohistochemistry for eNOS, iNOS, and nNOS

was performed using Universal LSAB Kits (Zymed

Histostain Plus Broad Spectrum, South San Francisco,

CA, USA) according to the manufacturer’s protocol.

Briefly, paraffin sections (5 mm) were treated with

primary antibodies for eNOS, iNOS, nNOS (Santa Cruz

Biotechnology, Santa Cruz, CA, USA) at 1:300

dilutions for 30 min at 37 8C. Afterwards, paraffin

sections were washed three times in phosphate buffered

saline (PBS) and incubated with biotinylated secondary

antibody for 30 min at room temperature (RT). Samples

were then washed three times in PBS and incubated in

streptavidin-HRP for 30 min at RT. Following rinsing in

PBS for 3 � 5 min, the sections were rinsed in distilled

water and incubated in 3,30-diaminobenzidine (DAB)

(Zymed Laboratories, San Francisco, CA, USA) for

5 min to reveal positive signals. Counterstaining was

carried out with haematoxylin. Sections were cleared in

xylene and mounted with Entellan. Negative controls

were performed using antibody dilutent solution instead

of the primary antibody. Relative immunoreactivity of

antibody staining was evaluated by two independent

observers in a blinded fashion and scored as follows: 0,

no immune reactivity; 1, weak immune reactivity; 2,

moderate immune reactivity; 3, strong immune reac-

tivity; 4, very strong immune reactivity.

2.4. Statistics

All values are presented as mean � S.D. For

statistical evaluation of the data, one-way ANOVA

was used. To compare the individual means of treatment

groups, the Tukey test was performed. Group differ-

ences were declared significant at P < 0.05.

3. Results

3.1. Uterine contractility

Electrically induced uterine smooth muscle con-

tractility of treatment groups in the presence of different

substances at various concentrations [L-arginine

A. Bulbul et al. / Theriogenology 68 (2007) 1156–1168 1159

Fig. 1. Recordings following L-arginine (10�5 M, 10�4 M, 10�3 M), SNP (10�8 M, 10�7 M, 10�6 M, 10�5 M) and 8-Br-cGMP (10�8 M, 10�7 M,

10�6 M) treatments and inhibition of EFS-induced uterine contractions (A: group Ov; B: group P; C: group E2; D: group E2 + P).

Fig. 2. Amplitude values of contractions in groups induced by EFS in

the presence of various concentrations of L-arginine, NNA + L-arginine,

and MB + L-arginine. Letters (a, b, c) indicate significant differences

between them in each column. P-value was <0.001 in groups Ov and

E2 + P, and <0.05 in group P, as determined by ANOVA.

(10�5 M, 10�4 M, 10�3 M), SNP (10�8 M, 10�7 M,

10�6 M, 10�5 M), and cGMP (10�8 M, 10�7 M,

10�6 M)] was recorded. Amplitude values and dose

responses are shown in Fig. 1.

The most effective dose of L-arginine in decreasing

uterus contractility was 10�3 M. Uterine contractile

tension due to L-arginine application significantly

decreased among the groups Ov (P < 0.01), P

(P < 0.05), and E2 + P (P < 0.01). None of the L-

arginine doses were effective at decreasing uterine

contractility in the E2 group. L-NNA (10�3 M) + L-

argininine (10�3 M) treatment decreased contractile

tension in all groups when compared to standard

contractile tension, though this difference was not

significant. Even though L-argininine (10�3 M) treat-

ment decreased the contractile tension, MB incubation

followed by L-argininine (10�3 M) treatment did not

alter the contractile tension in groups Ov, P, and E2 + P

(Fig. 2).

SNP application resulted in relaxation of uterine

contractile tension in the Ov (P < 0.001), P

(P < 0.001), and E2 + P (P < 0.01) groups. However,

none of the SNP doses were effective at decreasing

uterine contractility in the E2 group (Fig. 3). Similarly,

none of the cGMP doses were effective in the E2 group,

whereas cGMP caused a significant relaxation in the Ov,

P, and E2 + P groups (P < 0.05). The most effective

cGMP dose was 10�6 M (Fig. 4).

The percentage inhibition of contractility (when

contraction obtained by electrical field stimulation was

set as 100%) in the treatment groups is presented in

Table 2 and Figs. 5–7. L-Arginine (10�3 M), 8-Br-cGMP

(10�6 M), and all doses of SNP inhibited the contractile

tension of uterine smooth muscle (P < 0.001). Contrac-

tile tension was inhibited to a greater extent in the Ov and

P groups, followed by the E2 + P group. The least

affected treatment group was E2.

L-Arginine (10�3 M) applied to uterine tissue in the

presence of L-NNA did not cause any difference among

the groups. There was no difference between the P and

E2 + P groups following L-arginine treatment after

30 min of MB incubation, while MB inhibited the

contractility more in the Ov group than in the E2 and

E2 + P groups (P < 0.001).

A. Bulbul et al. / Theriogenology 68 (2007) 1156–11681160

Fig. 3. Amplitude values of contractions in groups induced by EFS in

the presence of various concentrations of SNP. Letters (a, b, c) indicate

significant differences between them in each column. P-value was

<0.001 in groups Ov and P, and<0.01 in group E2 + P, as determined

by ANOVA.

Fig. 4. Amplitude values of contractions in groups induced by EFS in

the presence of various concentrations of 8-Br-cGMP. Letters (a and b)

indicate significant differences between them in each column. P-value

was <0.05 in groups Ov, P and E2 + P, and <0.05 in group E2 + P, as

determined by ANOVA.

Table 2

Percentage inhibition of uterine muscle contraction in experimental groups with the presence of various concentrations of L-arginine, NNA + L-

arginine, and MB + L-arginine

Treatment Experimental groups

Ov (n = 10) P (n = 10) E2 (n = 10) E2 + P (n = 10) P<

L-arginine 10�5 6.50 � 1.09 a 6.20 � 0.84 a 0.80 � 0.24 b 5.20 � 0.55 a 0.001

L-arginine 10�4 13.65 � 3.01 a 11.80 � 1.50 a 3.35 � 2.14 b 9.80 � 0.91 a 0.01

L-arginine 10�3 33.85 � 3.48 a 31.19 � 3.06 a 1.85 � 6.78 c 22.70 � 1.64 b 0.001

L-NNA + L-arginine 10�3 14.94 � 6.40 2.90 � 3.96 0.50 � 6.98 5.40 � 3.83 b NS

MB + L-arginine 10�3 34.57 � 3.38 a 25.98 � 2.48 ab 2.53 � 6.67 c 19.87 � 2.41 b 0.001

SNP 10�8 19.00 � 2.94 a 16.30 � 2.55 a 0.80 � 0.25 c 6.00 � 0.68 b 0.001

SNP 10�7 28.80 � 2.71 a 23.30 � 3.09 a 1.10 � 0.27 c 9.30 � 0.93 b 0.001

SNP 10�6 38.30 � 3.53 a 36.40 � 3.16 a 1.70 � 0.61 c 16.10 � 1.29 b 0.001

SNP 10�5 60.10 � 2.52 a 56.20 � 4.11 a 2.70 � 0.58 c 24.70 � 1.76 b 0.001

8-Br-cGMP 10�8 6.60 � 0.60 a 4.90 � 0.79 b 0.80 � 0.25 c 7.70 � 0.57 a 0.001

8-Br-cGMP 10�7 16.30 � 1.98 a 13.80 � 0.99 ab 1.00 � 0.25 c 12.20 � 0.77 b 0.001

8-Br-cGMP 10�6 32.00 � 2.78 a 28.84 � 2.31 a 1.10 � 0.35 c 22.10 � 1.34 b 0.001

Ov = control, Ov rats injected intramuscularly (i.m.) with 0.2 ml sesame oil; E2 = Ov rats injected with 17b estrodiol (10 mg/(rat day) i.m.); P = Ov

rats injected with progesterone (2 mg/(rat day) i.m.); E2 + P = Ov rats injected with both 17b-estrodiol (10 mg/(rat day) i.m.) and progesterone

(2 mg/(rat day) i.m.). Letters (a, b, c) in the same line indicate significant differences between different letters. NS = not significant.

Fig. 5. Percentage inhibition of contractions in groups in the presence

of various concentrations of L-arginine (10�5 M, 10�4 M, 10�3 M),

NNA (10�3 M) + L-arginine (10�3 M), and MB (10�5 M) + L-arginine

(10�3 M) on uterine muscle.

Fig. 6. Percentage inhibition of contraction in groups in the presence

of various concentrations of SNP (10�8 M, 10�7 M, 10�6 M, 10�5 M)

on uterine muscle.

A. Bulbul et al. / Theriogenology 68 (2007) 1156–1168 1161

Fig. 7. Percentage inhibition of contraction in groups in the presence

of various concentrations of 8-Br-cGMP (10�8 M, 10�7 M, 10�6 M,

10�5 M) on uterine muscle.

3.2. Immunohistochemistry results (qualitative)

NOS expressions in uterus tissue sections are shown

in Figs. 8–13. The surface of glandular epithelium and

connective tissue cells from endometrium and myome-

trium showed various levels of nNOS, iNOS and eNOS

expression. Moreover, nNOS and eNOS expression was

present in nerve fibers. nNOS and iNOS expression was

detected in the endothelium of uterine vessels and in

Fig. 8. Representative images of nNOS immunostaining in endometrial surfa

in surface and glandular endometrial epithelium of the Ov group. (B) Modera

group. (C) Strong expression in surface and glandular endometrial epitheli

endometrial epithelium of the E2 + P group. Bar = 100 mm.

uterine smooth muscle. However, iNOS expression was

not observed in nerve fibers.

3.3. Immunohistochemistry scoring results (semi-

quantitative)

3.3.1. Neuronal NOS expression

Immunohistochemical scores for neuronal NOS

expression are listed in Table 3. There was no significant

difference in nNOS expression in the surface and

glandular endometrial epithelium between the Ov and P

groups, whereas expression significantly increased in

the E2 and E2 + P groups (P < 0.001). Moreover,

nNOS in the glandular epithelium was significantly

higher in the E2 + P group compared to the E2 group

(P < 0.001). nNOS expression was also observed in

myometrial smooth muscles and nerve fibers, with no

differences between groups.

3.3.2. Inducible NOS expression

Scores for inducible NOS expression are listed in

Table 4. The intensity of iNOS expression in

endometrial surface epithelium did not differ between

the Ov and P groups. However, iNOS expression was

greater in the E2 and E2 + P groups compared to the Ov

ce (arrow) and glandular (arrow head) epithelium. (A) Weak expression

te expression in surface and glandular endometrial epithelium of the P

um of the E2 group. (D) Strong expression in surface and glandular

A. Bulbul et al. / Theriogenology 68 (2007) 1156–11681162

Fig. 9. Representative images of nNOS immunostaining in myometrial smooth muscle cells and nerve fibers. (A) Weak expression in smooth muscle

cells and strong expression in nerve fibers in the Ov group. (B) Weak expression in smooth muscle cells and strong expression in nerve fibers in the P

group. (C) Moderate expression in smooth muscle cells and strong expression in nerve fibers in the E2 group. (D) Weak expression in smooth muscle

cells and strong expression in nerve fibers in the E2 + P group. Bar = 100 mm.

Fig. 10. Representative images of iNOS immunostaining in endometrial surface (arrow) and glandular (arrow head) epithelium. (A) Moderate

expression in surface and glandular endometrial epithelium of the Ov group. (B) Moderate expression in surface and glandular endometrial

epithelium of the P group. (C) Strong expression in surface and glandular endometrial epithelium of the E2 group. (D) Strong expression in surface

and glandular endometrial epithelium of the E2 + P group. Bar = 100 mm.

A. Bulbul et al. / Theriogenology 68 (2007) 1156–1168 1163

Fig. 11. Representative images of iNOS immunostaining in myometrial smooth muscle cells. (A) Moderate expression in myometrial smooth

muscle cells of the Ov group. (B) Moderate expression in myometrial smooth muscle cells of the P group. (C) Moderate expression in myometrial

smooth muscle cells of the E2 group. (D) Moderate expression in myometrial smooth muscle cells of the E2 + P group. Bar = 100 mm.

Fig. 12. Representative images of eNOS immunostaining in endometrial surface (arrow) and glandular (arrow head) epithelium. (A) Moderate

expression in surface and glandular endometrial epithelium of the Ov group. (B) Moderate expression in surface and glandular endometrial

epithelium of the P group. (C) Strong expression in surface and glandular endometrial epithelium of the E2 group. (D) Strong expression in surface

and glandular endometrial epithelium of the E2 + P group. Bar = 100 mm.

A. Bulbul et al. / Theriogenology 68 (2007) 1156–11681164

Fig. 13. Representative images of eNOS immunostaining in myometrial smooth muscle cells. (A) Weak expression in myometrial smooth muscle

cells of the Ov group. (B) Weak expression in myometrial smooth muscle cells of the P group. (C) Weak expression in myometrial smooth muscle

cells of the E2 group. (D) Weak expression in myometrial smooth muscle cells of the E2 + P group. Bar = 100 mm.

Table 3

nNOS expression scores in endometrial surface and glandular epithelium, and myometrial smooth muscle cells and nerve fibers, in all groups

Experimental groups

Ov (n = 10) P (n = 10) E2 (n = 10) E2 + P (n = 10) P<

Surface 1.20 � 0.20 a 1.75 � 0.17 a 3.00 � 0.25 b 3.16 � 0.16 c 0.001

Glandular 1.20 � 0.20 a 1.58 � 0.15 ab 1.83 � 0.10 b 2.66 � 0.21 c 0.001

Muscle 1.20 � 0.12 1.41 � 0.15 1.08 � 0.08 1.58 � 0.15 NS

Nerve fibers 3.20 � 0.20 3.50 � 0.22 3.00 � 0.25 3.50 � 0.22 NS

Ov = control, Ov rats injected intramuscularly (i.m.) with 0.2 ml sesame oil; E2 = Ov rats injected with 17b-estrodiol (10 mg/(rat day) i.m.); P = Ov

rats injected with progesterone (2 mg/(rat day) i.m.); E2 + P = Ov rats injected with both 17b-estrodiol (10 mg/(rat day) i.m.) and progesterone

(2 mg/(rat day) i.m.). Letters (a, b, c) in the same line indicate significant differences between different letters. NS = not significant.

and P groups (P < 0.001). iNOS expression in uterine

glandular epithelium of the Ov, P, and E2 groups was

not different, whereas iNOS expression was signifi-

cantly increased in the E2 + P group compared to the Ov

Table 4

iNOS expression scores in endometrial surface and glandular epithelium a

Experimental groups

Ov (n = 10) P (n = 10)

Surface 1.30 � 0.20 a 1.60 � 0.24 a

Glandular 1.60 � 0.24 a 1.40 � 0.24 a

Muscle 1.40 � 0.19 1.40 � 0.19

Ov = control, Ov rats injected intramuscularly (i.m.) with 0.2 ml sesame oil;

rats injected with progesterone (2 mg/(rat day) i.m.); E2 + P = Ov rats inje

(2 mg/(rat day) i.m.). Letters (a, b) in the same line indicate significant dif

and P groups (P < 0.05). There was moderate iNOS

expression in myometrial smooth muscle cells in all

groups. In addition, a variable, weak, and moderate

expression of iNOS was found in some stromal cells.

nd myometrial smooth muscle cells, in all groups

E2 (n = 10) E2 + P (n = 10) P<

2.60 � 0.24 b 3.20 � 0.20 b 0.001

2.00 � 0.24 ab 2.40 � 0.24 b 0.05

1.60 � 0.19 1.40 � 0.19 NS

E2 = Ov rats injected with 17b-estrodiol (10 mg/(rat day) i.m.); P = Ov

cted with both 17b-estrodiol (10 mg/(rat day) i.m.) and progesterone

ferences between different letters. NS = not significant.

A. Bulbul et al. / Theriogenology 68 (2007) 1156–1168 1165

Table 5

eNOS expression scores in endometrial surface and glandular epithelium, and myometrial smooth muscle cells and nerve fibers, in all groups

Experimental groups

Ov (n = 10) P (n = 10) E2 (n = 10) E2 + P (n = 10) P<

Surface 1.24 � 0.19 a 2.60 � 0.25 b 3.60 � 0.25 c 3.80 � 0.20 c 0.001

Glandular 1.36 � 0.37 a 2.60 � 0.25 b 3.40 � 0.25 c 3.80 � 0.20 c 0.001

Muscle 1.00 � 0.00 1.00 � 0.00 1.20 � 0.20 1.00 � 0.00 NS

Nerve fibers 3.00 � 0.00 3.00 � 0.00 2.900 � 0.10 3.00 � 0.00 NS

Vessel 0.40 � 0.25 0.60 � 0.25 0.60 � 0.25 0.80 � 0.20 NS

Ov = control, Ov rats injected intramuscularly (i.m.) with 0.2 ml sesame oil; E2 = Ov rats injected with 17b-estrodiol (10 mg/(rat day) i.m.); P = Ov

rats injected with progesterone (2 mg/(rat day) i.m.); E2 + P = Ov rats injected with both 17b-estrodiol (10 mg/(rat day) i.m.) and progesterone

(2 mg/(rat day) i.m.). Letters (a, b, c) in the same line indicate significant differences between different letters. NS = not significant.

3.3.3. Endothelial NOS expression

Immunohistochemical scores for endothelial NOS

expression are listed in Table 5. The intensity of eNOS

expression in endometrial surface and glandular

epithelium was greater in the E2 and E2 + P groups.

Moreover, eNOS expression was significantly higher in

group P than group Ov (P < 0.001). There was weak

expression in smooth muscle cells and vessel endothe-

lium layers in the myometrium in all groups.

4. Discussion

In rat serum, the physiological concentration of L-

arginine ranges from 0.2 to 3 mM [19], and it has been

observed that L-arginine completely inhibits in vitro

smooth muscle contractility at doses of 0.01–1 mM [9].

Moreover, L-argininine has been reported to decrease

myometrial contractile tension in pregnant humans and

rats [8] and in non-pregnant human myometrium in a

dose-dependent manner [20]. The relaxation effect of L-

arginine in the uterus decreases during labor, is further

diminished by postpartum day 1, and is completely

absent by postpartum day 2 [9]. Similarly, L-arginine

decreased uterine contractile tension in the Ov, P, and

E2 + P groups in the current study. However, none of the

L-arginine doses were effective in decreasing uterine

contractile tension in the E2 group. In addition, the

percentage inhibition of uterine contractile tension was

higher in the Ov and P groups than the E2 group with the

most efficient dose of L-arginine (10�3 M). Thus, the

results of the current study suggest that the inhibition of

uterine contractility by L-arginine was prevented by

estrogen, and that progesterone does not alter L-

arginine-modulated contractile tension.

In the present study, the effect of L-arginine on the

contractile tension of uterine smooth muscle at its most

effective dose (10�3 M) disappeared when it was

applied together with L-NNA. It has been previously

reported that the dose-dependent inhibition of smooth

muscle contractility by L-arginine is linked to nitric

oxide formation by NOS [8,9,20]. As L-NNA is a NOS

inhibitor, the above result of the current study suggests

that the relaxation of uterine smooth muscles caused by

L-arginine is mediated by nitric oxide.

SNP is an exogenous nitric oxide source, which

completely blocks spontaneous uterine contractility in

pregnant rats [9] and in non-pregnant humans [20].

Similarly, SNP completely removes spontaneous

myometrial contractility during pregnancy, but it is

not effective together with KCl [8]. Word and Cornwell

[21] have stated that SNP doses lower than 10�4 M are

not effective at inhibiting oxytocin-stimulated uterus

contractility, and higher doses cause irreversible

inhibition due to toxicity. In the current study, SNP

treatment at doses of 10�8 to 10�5 M inhibited

contractile tension in all groups except for the E2

group, with decreased inhibition in the E2 + P group.

Hence, it may be suggested that estrogen (but not

progesterone) prevents SNP-mediated uterine relaxa-

tion by SNP.

Nitric oxide conjugates and frees cyclic GC (cGC),

and hence increases cGMP levels and consequently

decreases uterine contractile tension [22]. Buhimschi

et al. [10] reported that the NO-cGMP pathway is

responsible for quiescence of the uterus in pregnant

women. Yallampali et al. [23] concluded that 17b-

estradiol and 17b-estradiol + progesterone treatment

decreases uterine cGMP and NO levels when compared

to control and progesterone-treated groups, and that

there is a positive correlation between NO formation

and cGMP levels. Krumenacker et al. [24] concluded

that 17b-estradiol inhibits soluble GC mRNA release in

uterine tissue. However, cGMP analogues do not inhibit

uterine contractility in non-pregnant monkey [25],

human [10,20] or mice [26]. Furthermore, nitric oxide-

driven inhibition of uterine contractility occurs inde-

pendently from the GC-cGMP pathway [10,25,26]. In

the current study, the most effective dose of cGMP was

A. Bulbul et al. / Theriogenology 68 (2007) 1156–11681166

10�6 M, and cGMP treatment of uterine tissues caused

relaxation in the Ov, P, and E2 + P groups. However, no

dose of cGMP was effective in the E2 group. This

suggests that progesterone had no effects on the cGMP

pathway. Estrogen, however, completely inhibited the

efficacy of cGMP-induced relaxation in uterine smooth

muscles.

It has been reported that MB inhibits cGC and

increases the contractile tension of smooth muscles

[27,28], while relaxation of spontaneous or induced

contractions mediated by the nitric oxide donor S-

nitroso L-cysteine (cys-NO) [29] and SNP [27,28] is

unaffected by prior addition of MB. In our study, we

showed that L-arginine-induced uterine contractility in

the Ov, P, and E2 + P groups decreased in presence of

MB. This is consistent with previous studies [20,29],

which suggested that NO-induced relaxation of the

myometrium occurs via a different pathway from the

cGC-cGMP pathway. It has also been suggested that

NO and/or cGMP may cause relaxation by directly

activating K+ channels in smooth muscles [30,31].

Modzelewska et al. [32] showed that diethylamine/NO

causes concentration-dependent decreases in amplitude

and frequency of myometrial contraction in non-

pregnant women, but that blockers of large-conduc-

tance and voltage-dependent Ca(2+)-activated K(+)

channels, including iberiotoxin and charybdotoxin,

reduce the diethylamine-nitric oxide-induced inhibition

of spontaneous activity of the myometrium.

NOS can moderate the motility effects of estrogen

and progesterone in the uterus. Progesterone regulates

iNOS expression in pregnancy, while estrogen receptors

regulate cNOS expression in the menstrual cycle [33].

In the current study, there was an intense nNOS

expression in nerve fibers between smooth muscle

layers of myometrium. Nevertheless, differences in

nNOS expression in nerve fibers in the uterus was not

significant among the groups. Similarly, Papka et al.

[34] reported nNOS expression in nerve fibers in the rat

uterus and suggested that nitric oxide-synthesizing

nerves in the rat myometrium might have a role in

controlling uterine vascularization. In addition, nNOS

expression in nerve fibers in smooth muscle layers has

been reported in the rabbit uterus, and that steroid

hormones had no effect on nNOS expression in nerve

fibers [35]. The results of the current study show that

there is weak nNOS expression in surface and glandular

epithelial cells and smooth muscle cells of the rat uterus,

except in myometrial nerve fibers of Ov rats. 17b-

Estradiol and particularly 17b-estradiol + progesterone

increased nNOS expression in the surface and glandular

epithelium of rat uteri. Similarly, Ogando et al. [36]

reported that there was no nNOS expression by western

blot analysis in uterine extracts of Ov rats, while nNOS

expression was observed in animals treated with

estrogen or progesterone.

In the present study, E2, P, and E2 + P treatment of

Ov rats significantly increased the intensity of eNOS

expression in surface and glandular epithelial cells,

while no significant difference was observed in smooth

muscle cells, nerve fibers, or endothelial cells. Some

studies have reported that estrogen [37,38] or proges-

terone [36,39] solely increase the intensity of eNOS

expression in uterine endometrial cells and smooth

muscle. In the current study, separate or combined

treatment of steroid hormones increased eNOS expres-

sion in surface and glandular epithelial cells, but only

weak expression was observed in all treatment groups.

Furthermore, estrogen has been reported to increase

eNOS expression in smooth muscle cells [40,41], and

Gangula et al. [42] reported that progesterone increases

in vitro eNOS in myometrial smooth muscle cells.

Contrary to the previous reports, our results suggest that

steroid hormones do not alter eNOS expression in

smooth muscle cells.

Huang et al. [43] reported weak iNOS expression

only in endometrial surface epithelial cells and mast

cells of Ov mice, and that progesterone treatment

increased iNOS expression in surface epithelial cells.

Yallampalli and Dong [41] have suggested that estradiol

reduce iNOS mRNA and protein levels in uteri of

pregnant and LPS-treated rats. However, in the current

study, there was a weak iNOS expression in endometrial

surface and glandular epithelium and myometrial

smooth muscle cells of rats. 17b-Estradiol increased

iNOS expression in surface epithelial cells of endome-

trium, and 17b-estradiol + progesterone treatment

increased iNOS expression in both surface epithelial

cells and glandular epithelial cells. Similarly, Saxena

et al. [13] reported that estrogen induces iNOS

expression, with greater expression when estrogen

and progesterone are applied together. Ogando et al.

[36] concluded that estrogen increases iNOS protein

expression in uteri of Ov rats. In addition, Huang et al.

[43] reported that undeterminable iNOS expression in

epithelial cells of Ov rats following estrogen treatment

becomes detectable after estrogen + progesterone treat-

ment.

In conclusion, the L-arginine–NOS–NO pathway

inhibits myometrial contractions via cGMP-dependent

and -independent mechanisms, and while progesterone

maintains the effects of nitric oxide, estrogen prevents

them. Furthermore, estrogen increases the expression of

nNOS, iNOS and eNOS enzymes in uterine epithelium

A. Bulbul et al. / Theriogenology 68 (2007) 1156–1168 1167

and glands. However, estrogen did not affect the

expression of the same enzymes in smooth muscles or

nerve cells and had no effect on uterine motility.

Acknowledgments

Financial assistance for this research was provided

by the Scientific Research Council of Turkey (project

numbered VHAG-2097). We are grateful to Dr Victor

M. S for his contributions.

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