Abstract

The aim of this study was to investigate the expression of RhoA/Rho-kinasein the uterus and the effect of Rhokinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n = 14, any procedure was not performed),vehicle group (n = 14, 0.2 ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n = 16, DHEA 6 mg/100 g in 0.2 ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCl, carbachol, and PGF2α induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCl, carbachol, and PGF2α induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCl, carbachol, and PGF2α induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rhokinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.

1. Introduction

Polycystic ovary syndrome (PCOS) affects 8.7%-17.8% of women in reproductive age group and it is associated with increased subfertilityinfertility risk in these women [1]. Since the etiopathogenesis of the disease remains still unknown, current treatment choices are symptomatically performed.Infertility observed in approximately 40% of women with PCOS is frequently associated with oligo-anovulation [2-4]. In these patients, spontaneous pregnancies are reported to occur in 67% of patients while ovulation induction treatments and assisted reproductive techniques are used to achieve pregnancy depending on the age and hormonal levels of the patients [5]. However, in these pregnancies, it was reported that spontaneous abortion, ectopic pregnancy, and preterm birth rates were high and it was suggested that these pregnancy complications were related to hyperandrogenism, hyperinsulinemia, insulin resistance and endometrial changes [6-8]. It is especially known that changes in the hypothalamic-pituitary-ovarian axis cause subfertility, infertility and early pregnancy losses in these patients. However, the information about the relationship between these clinical situations which may occur in PCOS and possible molecular and functional changes in uterus myometrium is inadequate.

Uterine contractions are achieved by the smooth muscle cells in the myometrium layer. The contraction mechanism in smooth muscle cells is organized with Ca2+-dependent and Ca2+-independent pathways [9,10]. Increased sensitization of uterine smooth muscles against Ca2+ ions is responsible for the Ca2+-independent pathway. This mechanism involves Rho/Rho-kinase pathway activation. The Rho proteins (from A to E and G isoforms) are small monomeric GTP-binding proteins and are the member of Rho subfamily that belongs to the Ras superfamily. The Rho-kinase is one of the effectors of Rho protein and has two isoforms called ROCK 1 and ROCK 2. Activated Rho activates ROCK by interacting with the Rho-binding domain (RBD) localized in a learn more C-terminal part of ROCK [11,12]. Rho/Rho-kinase pathway increases phosphorylation of myosin light chain (MLC) via decreasing myosin light chain phosphatase (MLCP) activity. Thus, in the smooth muscle cell, MLC phosphorylation increases with the low level of intracellular Ca2+ ion and the contraction mechanism is activated [12,13].

Previous studies reported that there were changes in uterine contraction and the expression of Rho/Rho-kinase during pregnancy and at the end of pregnancy [13–15]. However, to our knowledge, there has been limited report in the literature about the changes in uterine contraction in PCOS. Considering the ethical limitations in human studies, animal models such as dehydroepiandrostenedione (DHEA)-induced that reflect many features of PCOS are crucial resources to investigate this syndrome. Rodent models provide an important tool for interpreting the precise biological mechanisms associated with PCOS. There are numerous advantages of using rats such as ease of handling, maintenance and affordability, short estrous cycles and shorter reproductive lifespan [16,17]. Therefore, the aim of our study was to investigate the effect of Rho A/Rho-kinase pathway in uterine contraction changes in dehydroepiandrosterone (DHEA)-induced PCOS rats.

2. Materials and methods
2.1. Animals and experimental design

This study was approved by the Local Ethics Committee for Animal Experiments of the University of Mersin, Turkey (Project number: 2015/14). All the experiments were performed according to the approved National Institutional Guidelines for the Care and Use of Laboratory Animals.Forty-four female Sprague-Dawley rats (21 days old, average body weight 30–40 g) were supplied by the Experimental Animal Center, University of Mersin. The animals were kept in a lightcontrolled room under a 12 h/12 h light/dark cycle and controlled temperature (22–24 °C), 40–60% humidity, they were fed a pelleted electrodiagnostic medicine rat chow (MBD Animal Food Factory, Kocaeli, Turkey) and had access to water ad libitum. The animals were randomly divided into three groups. The control group (n = 14): any procedure was not performed on rats. The vehicle group (n = 14): 0.2 ml sesame oil (Sigma-Aldrich Corp., St. Louis, MO, USA) were injected subcutaneously to rats daily 20 consecutive days. PCOS group (n = 16): DHEA (EMD Millipore Corp., Billerica, MA, USA) (6 mg/100 g/day dissolved in 0.2 ml sesame oil) were injected subcutaneously to rats daily for 20 consecutive days in order to induce PCOS disease [18,19].At the end of the experiment, animals were anesthetized by intraperitoneal injections of 10 mg/kg xylazine hydrochloride (Rompun®, Bayer Türk Kimya Limited Şirketi, İstanbul, Turkey) and 90 mg/kg ketamine hydrochloride (Ketalar®, Pfizer İlaçları Limited Şirketi, İstanbul, Turkey) and sacrificed by cervical dislocation in estrous phase. Twenty-two rats (control, vehicle and PCOS groups, n:7, 7 and 8 respectively) were used for histological analysis (left uterine horns) and gene expression analysis (right uterine horns). Twenty-two rats (control, vehicle and PCOS groups, n:7, 7 and 8 respectively) were used for isolated organ bath experiments.

2.2. Tissue collection

For histologic analysis, the ovaries and the left uterine horns were fixed in 10% neutral buffered formalin (NBF) for 24 h. For gene expression analysis, the right uterine horns were frozen in liquid nitrogen and stored at −80 °C until total RNA extraction. For isolated organ bath experiments, bilateral uterine horns were placed in Krebs solution.

2.3. Vaginal smears and estrous cycle determination

Phases of the estrous cycle (proestrous, estrous metestrous and diestrous) were determined in the vaginal smears. Two weeks after the start of the experiment, vaginal smears were taken from all rats (35 days old) on daily basis at 9:00-10:00 am. Each rat’s phase of the estrous cycle (proestrous, estrous metestrous and diestrous) was monitored and evaluated for 10 days and the rats had two estrous cycles of 4–5 days included in the study. Then rats were killed at the estrous phase of the third estrous cycle. The vaginal smear samples stained with crystal violet and observed under a light microscope (Olympus BX50, Olympus Corporation, Tokyo, Japan). The phase of estrous cycle was determined as follows [20]: proestrus phase (small, round to oval, nucleated epithelial cells; parabasal cells), estrus phase (anucleated and cornified epithelial cells), metestrus phase (anucleated epithelial cells, nucleated epithelial cells, and leukocytes), and diestrus phase (a high number of leukocytes).

2.4. Ovarian morphology and myometrium thickness

After fixation, the tissues were dehydrated, cleared and embedded in paraffin. Tissue sections (5 μm thick) were cut by microtome and stained with hematoxylin-eosin (H&E). Stained sections were examined and photographed by a light microscope (Olympus BX50, Olympus Corporation, Tokyo, Japan) equipped with a digital camera (Olympus LC30, Olympus Corporation, Tokyo, Japan). The myometrium thickness was measured using LCmicro image analysis software (Olympus Corporation, Tokyo, Japan) in 4 different areas of each section and mean myometrium thickness was calculated for each uterus.

2.5. Immunohistochemistry

Formalin-fixed uterine horns were dehydrated, cleared and embedded in paraffin. Five-micrometer sections were cut from paraffinembedded tissue blocks on to poly-L-lysine coated slides. Sections were deparaffinized, rehydrated and heated in citrate buffer (pH: 6.0, 98 °C, 20 min) for antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Non-specific binding sites were blocked with blocking solution (Mouse and Rabbit Specific HRP Plus Detection IHC Kit, Abcam, Cambridge, MA, USA) for 10 min. Afterwards, the sections were incubated overnight at 4 ºC with a rabbit polyclonal primary antibody against Rho A (1:300, NB100-91273, Novus Biologicals, Littleton, CO, USA), rabbit monoclonal primary antibody against ROCK 1 (1:100, ab45171, Abcam, Cambridge, MA, USA) and rabbit polyclonal primary antibody against ROCK 2 (1:200, ab137697, Abcam, Cambridge, MA, USA). Negative control sections were incubated with a phosphate buffered saline-bovine serum albumin (PBS-BSA) instead of the primary antibody. The sections were incubated with a biotinylated goat anti-polyvalent secondary antibody (Mouse and Rabbit Specific HRP Plus Detection IHC Kit, Abcam, Cambridge, MA, USA) for 10 min and then they were incubated in a streptavidin-peroxidase (Mouse and Rabbit Specific HRP Plus Detection IHC Kit, Abcam, Cambridge, MA, USA) for 10 min.For Rho A, ROCK 1 and ROCK 2 immunoreactivity, four different areas from the uterus of each rat were evaluated by a combinative semiquantitative scoring system. The immunoreactivities in myometrium smooth muscle cells were determined with the immunoreactive score (IRS). The IRS was given a range of 0– 12 as a result of multiplication between positive cells proportion score (0–4) and staining intensity score (0–3) (Table 1) [21].

2.6. RNA isolation and quantitative real-time PCR

The tissue samples were homogenized by use of MagNA Lyser Green Beads (Cat. No: 03358941001, Roche Diagnostics, Penzberg, Germany). Total RNA was extracted using the High Pure RNA Tissue Kit (Cat. No: 12033674001, Roche Diagnostics, Penzberg, Germany). RNA concentration and purity were measured with a NanoQ microvolume spectrophotometer (CapitalBio Technology, Beijing, China). RNAs were reverse transcribed using the Transcriptor First Strand cDNA Kit (Cat.No: 04896866001, Roche Diagnostics, Penzberg, Germany) in a PrimeG Thermal Cycler (Techne Instruments, Cambridge, UK). Real-Time Ready Catalog Assays used in quantitative PCR were as follows: Rat Rho A Real-Time Ready Catalog Assay (Cat no: 05583055001, Roche Diagnostics, Penzberg, Germany), Rat Rock 1 Real-Time Ready Catalog Assay (Cat no: 05532957001, Roche Diagnostics, Penzberg, Germany), Rat Rock 2 Real-Time Ready Catalog Assay (Cat no: 05532957001, Roche Diagnostics, Penzberg, Germany), Rat ACTB (beta-actin) Gene Real-Time Ready Catalog Assay (Cat no: 05532957001, Roche Diagnostics, Penzberg, Germany). The PCR reaction (20 μL) included 5 μL cDNA template, ready primer-probe 1 μL, 10 μL master mix. Incubation was conducted for 95 °C for 10 min for the activation of DNA polymerase. The amplification parameters were 45 cycles of 95 °C for 10 s, followed by 60 °C for 30 s and 72 °C for 1 s of elongation. Relative mRNA expression was analyzed by using the comparative Ct (ΔΔCt) method where the relative expression is calculated as 2 −ΔΔCt and normalized respect to the reference gene, beta-actin. All analyses were performed in triplicate.

Fig. 1. Ovarian morphology in control group (A), vehicle group (B) and PCOS group (C) (H&E, ×40). Normal ovarian morphology is shown in control group and vehicle group (asterisks: corpora luteum, Nf: normal follicle). A few cystic follicles (Cf), numerous atretic follicles (arrowhead), and some normal follicles (Nf) are shown in the ovary of the PCOS group (C).

2.7. Isolated organ bath experiments

The uterus was immediately isolated and adherent tissues were carefully removed in a petri dish, containing Krebs solution (composition in mM: NaCl 118, KCl 4.8, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11, and Na2EDTA 0.01). Four strips (approximately 1 mm wide and 1 cm long) were prepared from one rat uterus. The weight of uterine strips was measured and mean weight of uterine strip was calculated for each rat.The uterine strips were suspended in organ baths filled with Krebs’ solution (37 °C) and gassed with 95% O2 and 5% CO2 under 1 g of initial tension. Tissue responses were recorded isometrically with a force transducer (COMMAT, Ankara, Turkey) and displayed on a Biopac acquisition system (Biopac Systems, Goleta, CA, USA). The strips were allowed to equilibrate at optimum resting tensions for 60 min during which time the bath was replaced with a fresh Krebs solution every 20 min. Following equilibration, the uterine strips were contracted using 80 mM KCl (dissolved in distilled water) and after a steady state of contraction was obtained, the tissues were washed and allowed to rest for 45 min more. Thereafter tissues were contracted with cumulative doses of KCl (10– 100 mM), carbachol (10 − 9-10-5 M) or PGF2α (10 − 9-10-5 M). Subsequently, the strips were washed and incubated with two rho-kinase inhibitors either fasudil (10-5 M) or Y-27632 (10-5 M) for 45 min and responses to the same contractile agents have obtained again. The contractions were either expressed as gram-force or normalized with wet tissue weight and expressed as mN/g-wet tissue weight. All drugs and chemicals that haven’t been stated otherwise were purchased from Sigma (Sigma Chemical Company, St. Louis, MO, USA).

2.8. Statistical analysis

Statistical analyses were performed using Graph Pad 3.0 (GraphPad Software, San Diego, CA) for Windows. Distributions of data were analyzed with the Shapiro-Wilk test. Normally distributed data were analyzed with parametric tests while not normally distributed data were analyzed with non-parametric tests. Kruskal-Wallis test and Dunn’s post hoc test were used for the comparison of mRNA expression levels variable. One-way analysis of variance (ANOVA) and Bonferroni post hoc test were used for other data. All data were presented as mean ± SEM. Statistical significance was determined as p ≤ 0.05.

3. Results
3.1. Ovarian morphology and myometrium thickness

The normal histological morphology was observed in the control group (Fig. 1A) and the vehicle group (Fig. 1B). The normal follicles at different stages of maturation and a few corpora luteum were observed without cystic follicles or other structural abnormalities in both groups. In PCOS group, the follicles at different stages of maturation, numerous atretic follicles, cystic follicles of different sizes were observed without corpora luteum (Fig. 1C).The uteri of the control group (Fig. 2A) and vehicle group (Fig. 2B) exhibited a thinner layer of myometrium than those in the uteri of PCOS group (Fig. 2C). Myometrium thickness significantly increased in the PCOS group (438.7 ± 35.92 μm) compared to the control group (292.1 ± 19.13 μm), vehicle group (303.6 ± 16.81 μm) (p < 0.01 and p < 0.01, respectively) (Fig. 2D).

3.2. Immunohistochemistry

The negative control sections showed no immunostaining (Fig. 3).Rho A, ROCK 1 and ROCK 2 immunostaining were observed in myometrial smooth muscle cells in the control group, vehicle group, and PCOS group (Fig. 3). The immunoreactive scores of Rho A, ROCK 1 and ROCK 2 in control group (6.42 ± 0.36, 5.57 ± 0.42 and 6.14 ± 0.40 respectively), vehicle group (6.14 ± 0.50, 5.28 ± 0.42 and 6.42 ± 0.57 respectively) and PCOS group (6.62 ± 0.56, 5.37 ± 0.46 and 6.25 ± 0.52 respectively) were similar and there were no statistically significant differences (all
comparisons p > 0.05, Fig. 4).

Fig. 2. The myometrium thickness (My) of uteri in the control group (A), vehicle group (B) and PCOS group (C) (H&E, ×40). Myometrium thickness was significantly increased in PCOS group when compared to control group and vehicle group (D) (data shown as mean ± SEM, One-way ANOVA with Bonferroni post hoc tests, ** p < 0.01 and p < 0.01, respectively).

3.3. Quantitative real-time PCR

mRNA expression levels of Rho A, ROCK 1 and ROCK 2 in control group (4.71 ± 0.80, 2.79 ± 0.68 and 3.39 ± 0.79 respectively), vehicle group (4.54 ± 1.08, 1.73 ± 0.59 and 3.42 ± 0.90 respectively) and PCOS group (6.86 ± 0.76, 1.92 ± 0.24 and 1.72 ± 0.19 respectively) were similar and there were no statistically significant differences (all comparisons p > 0.05, Fig. 4).

Fig. 3. The protein expression of Rho A, ROCK 1 and ROCK 2 in the myometrial smooth muscle cells and negative control sections (Indirect peroxidase, ×100). The immunostaining of Rho A, ROCK 1 and ROCK 2 was similar in all groups.

Fig. 4. The mRNA expression levels and the immunoreactive scores of Rho A, ROCK 1, and ROCK 2. The mRNA expression levels and the immunoreactive scores of Rho A, ROCK 1, and ROCK 2 were not statistically significantly different in uterine tissue of the PCOS group compared to the control group and vehicle group (data shown as mean ± SEM, Kruskal-Wallis test with Dunn’s post hoc tests and One-way ANOVA with Bonferroni post hoc tests, all comparisons p > 0.05).

3.4. Wet tissue weight and isolated organ bath experiments

Mean weight of uterine strips were 10.76 ± 0.50 mg, 10.94 ± 0.52 mg, 14.21 ± 0.88 mg in the control group, vehicle group, and PCOS group, respectively. In the PCOS group, uterine strip weight significantly increased when compared to the control group and vehicle group (p < 0.01 and p < 0.01, respectively) (Fig. 5).Contractile responses to cumulative doses of KCl (10– 100 mM), carbachol (10 − 9–10-5 M) or PGF2α (10 − 9–10-5 M) were not changed in the control group compared to the vehicle group (Fig. 6A–F). However contractile responses to cumulative doses of KCl (10– 100 mM) in PCOS group significantly increased when compared to the control group (Fig. 6A and B, p < 0.05–0.001). While contractions to lower doses (10 − 9-10-8 M) of carbachol significantly decreased, higher doses (106–10-5 M) were significantly increased (Fig. 6C and D, p < 0.05–0.01). Responses to PGF2α (10 − 9–10-5 M) in PCOS group were significantly increased when contractions expressed as pure gram-force without any normalization (Fig. 6E, p < 0.05). However, this significance was disappeared when contractions were normalized with wet tissue weight and expressed as mN/g (Fig. 6F). Also, the degree of significance of increased contractions in the PCOS group for KCl and carbachol were decreased when contractions normalized with wet tissue weight (Fig. 6A–D). This discrepancy might be due to the increase of wet tissue weight in the PCOS group.

Incubation of strips with two rho-kinase inhibitors either fasudil (10 − 5 M) or Y-27632 (10 − 5 M) for 45 min significantly inhibited KCl (10– 100 mM) induced contractile responses in control group, vehicle group and PCOS group (Fig. 7, p < 0.05–0.01).Incubation of strips with two rho-kinase inhibitors either fasudil (10 − 5 M) or Y-27632 (10 − 5 M) for 45 min significantly inhibited carbachol (10-9–10 − 5 M) induced contractile responses in all groups (Fig. 8, p < 0.05–0.01).Although limited significant inhibition was observed with fasudil (10 − 5 M) incubation only for 10 − 5 M PGF2α, there was a more significant inhibition for other doses of PGF2α in all groups with Y-27632 (10 − 5 M) which is a more potent inhibitor of Rho-kinase (Fig. 9,p < 0.05).

Fig. 5. Mean weight of uterine strips in control group, vehicle group, and PCOS group. Mean weight of uterine strip significantly increased in PCOS group when compared to control group and vehicle group (data shown as mean ± SEM, One-way ANOVA with Bonferroni post hoc tests, ** p < 0.01 and p < 0.01, respectively).

4. Discussion

In the present study, the relationship between uterine contraction changes and Rho A/Rho-kinase (ROCK 1 and ROCK 2) pathway which plays an important role in the smooth muscle contraction was investigated in the Sprague Dawley rat model of PCOS by histological, molecular, and functional methods.Many data were published concerning an increased rate of infertility and pregnancy complications such as spontaneous miscarriage and premature delivery that associated with functional and morphological changes in the endometrium due to the impaired hypothalamic-pituitary-ovarian axis in women with PCOS when compared to the normal population [2-4,6,8]. With regard to the functional role of myometrium, the clear association between PCOS and changed uterine contractions has not been fully elucidated. However, in addition to endometrial changes as well as the myometrial changes may be important for the increased rates of infertility and pregnancy complications in the PCOS. In a study on human myometrium with the magnetic resonance imaging method, it was reported that the uterus peristaltic activity changed in patients with PCOS compared to the control group [22]. Another study has reported that irregular uterine contractions and different mechanical responses of an isolated uterus in PCOS rats [23]. Previous studies have demonstrated that hyperandrogenised rats [24] and mice [25] with DHEA show significant increases in myometrium thickness and myometrium area. In agreement with these results, we observed that myometrium thickness in the PCOS group was significantly increased compared to the control group and the vehicle group. The increased myometrial thickness may be related to the proliferation of smooth muscle cells and/or amount of extracellular matrix component such as sulfated glycosaminoglycans in the myometrium layer [26]. In addition, previous studies have demonstrated that androgens promote the growth and differentiation of the rat uterus and cause increased myometrial thickness [24,27].Sajadi et al. [23] demonstrated that, after exposure to carbachol and oxytocin, uterine contractions were more irregular in the PCOS compared to control rats. The author suggested that increased myometrium thickness may be related to uterine contraction changes in PCOS. In accordance with this study, we determined that KCl, carbachol, and PGF2α-induced uterine contractions increased in PCOS.

Fig. 6. Cumulative dose-response curves of contractile agents. Responses to KCl (10-100 mM,A, B), carbachol (10 − 9-10-5 M, C, D), and PGF2α (10 − 9-10-5 M, E, F) were similar for the control group and vehicle group. However, Falsified medicine responses to KCl (A, B) were significantly increased in PCOS group. While contractions to lower doses (10 − 9-10-8 M) of carbachol significantly decreased, higher doses (10-6-10-5 M) were significantly increased (C, D). Responses to PGF2α in PCOS group were significantly increased when contractions expressed as gram-force (E), but this significance was disappeared when contractions were normalized with wet tissue weight and expressed as mN/gram (F) (data shown as mean ± SEM, One-way ANOVA with Bonferroni post hoc tests, * p < 0.05, ** p < 0.01, *** p < 0.001).

Fig. 7. The effect of Rho-kinase inhibitors, fasudil (left column) and Y-27632 (right column) incubation on KCl-induced contractions. Incubation of strips with two rho-kinase inhibitors either fasudil (10 − 5 M) or Y-27632 (10 − 5 M) for 45 min significantly inhibited KCl (10-100 mM) induced contractile responses in control group, vehicle group, and PCOS group. The contractions were normalized with wet tissue weight and expressed as mN/gr-wet tissue weight (data shown as mean ± SEM, One-way ANOVA with Bonferroni post hoc tests, * p < 0.05, ** p < 0.01, *** p < 0.001).

One of the important pathway contributing to uterine smooth muscle contractions is the Rho/Rho-kinase pathway. In different studies that have been performed in the uterus of humans [13,14] and animals [10,15], the relationship between the Rho/Rho-kinase pathway and uterine contraction of the non-pregnant, at the end of pregnancy and during the labour has frequently been reported. However, in the literature, there is no knowledge regarding the relationship between the uterine contraction changes in PCOS and Rho A/Rho-kinase pathway. Previous studies demonstrated that the contractile agents induced contractions were reduced by Rho-kinase inhibitors despite no change in expression of Rho A/Rho-kinase in the vascular system of rats [28] and mice [29]. The authors suggest that these results may be related to the augmented activation of Rho-kinase. The activity of Rho A/Rhokinase pathway is regulated by various positive and negative regulatory proteins. One of positive effect regulatory protein on the Rho A/Rhokinase pathway is Rho guanine nucleotide exchange factors (RhoGEFs) which stimulate the exchange of GDP for GTP and therefore increase the Rho protein activity [30]. Previous studies showed the activation effects of the RhoGEFs on the Rho A/Rho-kinase pathway in human myometrium [31] and rat pulmonary artery smooth muscle [32]. Similarly, Lartey et al. [33] showed that Rho A, ROCK 1, and ROCK 2 expressions did not change in non-pregnant, pregnant, and during the labour in human myometrium. They suggested that although Rho A, ROCK 1, and ROCK 2 expressions did not change, the decreased uterine contractions during pregnancy compared to term pregnant women were associated with the increase in the expression of the Rho family GTPase 2 (RND) 2 and Rho family GTPase 3 (RND 3) proteins, which have a negative regulatory effect on the Rho A/Rho-kinase pathway [33]. In the present study, we could not find a significant difference in mRNA expression levels and immunoreactive scores of Rho A, ROCK 1 and ROCK 2, although Rho-kinase inhibitors suppressed contractile agents induced uterine contractions in PCOS. However, one limitation of the present study was that we did not measure activation of Rho A/Rhokinase pathway.

When contractions evaluated without any normalization and expressed as pure gram-force, increased uterine contractions in PCOS were more pronounced. However, these increased contractions were disappeared for PGF2α and degree of increasement were reduced for KCl and carbachol when contractions were normalized with wet tissue weight and expressed as mN/g. This discrepancy might be due to the increase of wet tissue weight and uterine thickness in PCOS.It has been reported that both receptor agonist-triggered stimulation and depolarization with high KCl induce substantial Rho activation and Rho-kinase dependent contraction of smooth muscle [34]. Membrane depolarization with KCl activates the L-type of voltage-dependent calcium channels and stimulates the entry of extracellular calcium into the cell interior which is also responsible for Rho activation [35,36]. In our study, Rho-kinase inhibitors suppressed both agonist (carbachol and PGF2α) and KCl induced contractions of uterine smooth muscles in PCOS.

Fig. 8. The effect of Rho-kinase inhibitors, fasudil (left column) and Y-27632 (right column) incubation on carbachol-induced contractions. Incubation of strips with two rho-kinase inhibitors either fasudil (10 − 5 M) or Y-27632 (10 − 5 M) for 45 min significantly inhibited carbachol (10-9-10 − 5 M) induced contractile responses in control vehicle and PCOS groups. The contractions were normalized with wet tissue weight and expressed as mN/gr-wet tissue weight (data shown as mean ± SEM, One-way ANOVA with Bonferroni post hoc tests, * p < 0.05, ** p < 0.01, *** p < 0.001).

It has been reported that although both M2 and M3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M3 receptor [37]. In addition, anticholinergic agent hyoscine bromide was used to decrease the frequency of uterine peristalsis and thus facilitate retention of embryos and increase the probability of successful pregnancy within vitro fertilization [38]. Also, it has been reported that Y-27632, a Rho-kinase inhibitor, significantly attenuated the cholinergic agent carbachol-induced contraction without changing calcium levels in rat myometrium [39]. In the present study cholinergic contractions with carbachol were significantly decreased by Rho-kinase inhibitors, fasudil and Y-27632 both in PCOS and other groups tested. Also an interesting and so far has not been reported finding of the present study was that contractions to lower doses (10 − 9-10-8 M) of carbachol were blunted while higher doses (10-6-10-5 M) were still increased in myometrium of PCOS. This blunted cholinergic response is in accordance with the decreased relaxation obtained with acetylcholine via cholinergic M3 receptors in arteries of murine PCOS model [40,41]. However, further investigations are needed to fully elucidate blunted response to cholinergic stimulation in myometrium of PCOS.

Prostaglandins appear as primary regulators of uterine functions during the reproductive functions [42]. It has been reported that lots of PCOS-involved genes are perturbed by alprostadil (PGE1) and dinoprost (PGF2α) [43]. It was found that pro-inflammatory and vasoconstrictor PGF2α level is increased in DHEA-treated mice [44]. In addition, PGF2α has been shown to induce calcium sensitization in both human [45] and mouse [46] myometria. In our study, although both Y-27632 and fasudil inhibited the PGF2α induced contractions, this suppressive effect was more effectively observed by Y-27632. As there is evidence for higher serum level of PGF2α in PCOS, and that was not present in the organ bath, the question emerges, whether the interaction of the calcium-sensitizing prostaglandin and altered M3-receptor mediated response could be one component of altered uterine functions [44] Rho-kinase inhibitors are tested for a variety of diseases such as cardiovascular and neurovascular diseases [47-49]. In addition, fasudil has been suggested a promising agent for the treatment of endometriosis [50]. One of important prediction of the present study is that the decrease in uterine contractions could be seen as a side effect while using Rho-kinase inhibitors for other therapeutic strategies such as hypertension, atherosclerosis and cerebral vasospasm.

In conclusion, our findings show that uterine weight, myometrium thickness,and KCl, carbachol, and PGF2α-induced uterine contractions increased, although mRNA and protein expressions of Rho A, ROCK 1, and ROCK 2 did not change in PCOS. In addition, according to our contraction data, there seems to be no change of Rho-kinase pathway. Interestingly Rho-kinase inhibitors are capable of inhibiting increased uterine contractions in PCOS. Thus we suggest Rho-kinase inhibitors as potential therapeutic agents for uterine hypercontraction in PCOS.

Fig. 9. The effect of Rho-kinase inhibitors, fasudil (10− 5 M, left column) and Y-27632 (10 − 5 M, right column) incubation on PGF2α-induced contractions. Although limited significant inhibition was observed with fasudil (only for 10− 5 M PGF2α) incubation, there was more significant inhibition also for other doses of PGF2α in all groups with Y-27632. The contractions were normalized with wet tissue weight and expressed as mN/gr-wet tissue weight (data shown as mean ± SEM, One-way ANOVA with Bonferroni post hoc tests, * p < 0.05).