The endometrium functions to facilitate the establishment and maintenance of pregnancy. Preparation of a receptive endometrium is an important factor for successful implantation of the embryo. The endometrium is mainly composed of epithelia, stroma, leukocytes, and associated vascular elements. Uterine endometrial stroma undergoes extensive remodeling at the initial phases of the implantation process . These stromal connective tissue changes are considered as a key event for success of the implantation process. The endometrial stroma quite resembles the mesenchymal tissue. It is formed of an intermingled delicate framework of reticular fibers. In this framework, irregular stellate cells and extracellular matrix are deposited . Endometrial changes that are known to associate the implantation process include edema of the stroma [3–10], disruption of the collagen fibrils, and alteration in glycosaminoglycans (GAGs) that are thought to be the major polysaccharide components of the uterine extracellular matrix .
The major collagens of uterine stromal extracellular matrix are the fibrous collagen types I, III, and V and the basement-membrane-associated microfibril collagen type VI [11,12]. Hyaluronic acid (HA), a nonprotein-linked GAG, is a prominent component of the GAGs, particularly in rapidly growing and remodeling tissues . HA is a nonsulfated linear GAG that exists as a high-molecular weight polymer. It has a strong negative charge that attracts a large associated volume of water leading to voluminous expansion of tissues [13,14]. CD44 glycoprotein is the receptor for the HA .
Large-scale evidence suggests that prostaglandins (PGs), especially prostaglandin E2 (PGE2), are important modulators of events at the site of implantation in laboratory rodents [16–18]. PGs induce decidualization when instilled into a rat uterus primed for implantation [19–21]. PGE2 synthesis by early pregnant uterus increases at the time of implantation in rat . Concentrations of PGE2 in the early pregnant uterus are higher at the implantation site than in surrounding areas . Application of an artificial stimulus to induce decidualization in the rat or mouse uterus, results in a rapid rise in the uterine concentrations of PGE2 . However, the exact mechanism and pathway of action of PGE2 are not yet fully understood.
PGE2 mediates its effects by binding to four subtypes of receptors designated as EP1, EP2, EP3, and EP4 [25,26]. EP2 and EP4 receptors activate adenylate cyclase and lead to increased levels in intracellular cyclic AMP. EP1 activation is associated with an increase in intracellular Ca2+, and EP3 inhibits intracellular cyclic AMP levels. Utilizing synthesized PGE2 receptor agonists is a useful tool for studying PGE2 mechanism of action. Owing to the extreme importance of the endometrial connective tissue changes that associate with pregnancy, this study was undertaken to investigate the impact of PGE2 and its EP4 receptor agonist (EP4A) on selected endometrial elements commonly modified with initiation of pregnancy (collagen I, HA, GAGs, and CD44 molecule). Morphological and immunohistochemical analyses were carried out.
Materials and methods
Animals and drug treatments
To avoid confusion with the physiological events and the influence of steroids that change uterine composition, such as endometrial stromal collagen and GAGs across the estrous cycle and during pregnancy, immature rats were chosen for this study.
Immature female albino rats aged 22 days with approximately 50 g body weight were used in the study. Animals were purchased from the Research Unit and the Bilharzial Research Center of Faculty of Medicine (Ain Shams University, Cairo, Egypt). Rats were maintained under routine conditions with free access to food, water, and 12 h light:12 h darkness.
Rats were divided into three groups (10 animals/group). Group 1 (control group), where the rats received a single injection of 0.5 ml of normal saline. Group 2 (PGE2 group), where the rats received a single injection of 50 μg of PGE2 (1 mg/kg body weight)  dissolved in 0.5 ml of saline (Sigma-Aldrich, St Louis, Missouri, USA). Group 3 (EP4A group), where the rats received a single injection of 50 μg of EP4A (1 mg/kg body weight)  dissolved in 0.5 ml of saline (APS-999 Na, Toray Industries, Inc., Tokyo, Japan).
Drug injections were administered by the subcutaneous route. Rats were killed after 24h with a lethal dose of ether. The weight of the extracted rat uteri was immediately recorded. According to the fixative used, uteri specimens were divided into two groups.
The first group of specimens was fixed in 4% paraformaldehyde in phosphate buffered saline (pH 7.4). The second group was fixed in Carnoy's fixative (chloroform, acetic acid, and methanol at 30 : 10 : 60 (ml, respectively) for optimum fixation of HA owing to the crucial role of the fixation process in preserving HA. Specimens were processed for embedding in paraffin and 4-μm thick sections were obtained. Paraffin sections were stained with hematoxylin and eosin and Alcian blue (pH of 2.5)  for endometrial stromal total GAGs.
A computer-assisted image analysis system was used to analyze the stained slides. Light microscopic images of the specimens were captured and then transformed into 32-bit color images. For transformation, a digital camera (Fujix HC-2000; Fuji Photo Film, Tokyo, Japan) attached to a light microscope (Vanox AHBS3; Olympus, Tokyo, Japan; ×40 objective) was used.
Assessment of cellular spacing (quantification of cells)
For studying endometrial stromal edema, counting of fibroblast nuclei in the stroma was used as a means of assessment of cellular spacing in control and injected rats. Counting was performed in three ×200 power fields (0.7 mm2) by two independent observers (blinded to the specimen details), and the mean of the observations was calculated to reflect the density of cells. Results are expressed as mean+standard deviation.
- (1) Rabbit polyclonal anticollagen type I antibody with species reactivity to human, rat, and mouse (Sigma-Aldrich) was used at an optimal dilution of 1 : 2000;
- (2) Histological staining for HA was performed using a highly specific, biotinylated HA-binding peptide (bHABP), derived from a tryptic digest of bovine aggrecan, the chondriotin sulfate-rich proteoglycan of cartilage, and isolated by affinity chromatography using a column of HA-sepharose [13,14]. bHABP was purchased from Sigma Chemical Co.. The optimal dilution was 1 : 200;
- (3) Monoclonal mouse anti-CD44 antibody (Dako, Copenhagen, Denmark) was used at an optimal dilution of 1 : 50.
Immunohistochemical analysis was carried out using the streptavidin–biotin–peroxidase complex method . Collected uterine specimens were immediately embedded in optimal cutting temperature compound (Miles Inc., Indiana, USA) and frozen at −70°C. Cryostat sections of 6-μm thickness were prepared and air-dried for 1–2 h at room temperature. Slides were incubated with 0.3% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. After washing, they were treated with normal goat serum for 30 min to block nonspecific binding. Primary antibodies were then applied at the appropriate dilutions and incubated in 2% bovine serum albumin (Sigma) in phosphate buffered saline overnight at 4°C. After washing, slides were incubated with biotinylated secondary antibody for 45 min (except for HA staining in which bHABP was used), followed by incubation with the avidin–biotin–peroxidase complex (Vectastatin kit, Burlingame, California, USA). Reaction products were visualized after incubation with 0.025% diaminobenzidine and 0.003% hydrogen peroxide. Nuclear staining was performed with hematoxylin. Slides were dehydrated and mounted. Positive staining was recognized as a brown color. Negative control staining for each test was performed after omitting the primary antibody.
Intensity of immune reaction was evaluated using a semiquantitative subjective scoring system with a three-point score from – to ++ (–, absent; +, weak; ++, strong).
The data were analyzed using the statistical package for social sciences (SPSS, IBM SPSS, The Analytic Professional, USA) program. Conventional statistical procedures for comparison (Student's t-test) were used to evaluate the differences concerning the uterine weight and the stromal fibroblast nuclear count. The probability level of P value less than 0.05 was considered statistically significant.
Estimation of the uterine weight (mean±standard deviation) in the three test groups revealed a significant increase in weight in the PGE2 and EP4A groups [0.20±0.16996 (P=0.00078); 0.22±0.184797 (P=0.00089), respectively] in comparison with the control group (0.11±0.09592) (Histogram 1).
Light microscopic study
Hematoxylin and eosin staining
In the control group, the endometrium showed lining epithelium formed of a single layer of columnar epithelium resting on a layer of connective tissue stroma into which uterine glands extend (Fig. 1a and b). The stroma in sections of PGE2 and the EP4A groups (Figs 2 and 3) appeared more voluminous, and vascular channels were seen apparently more dilated compared with control group.
Quantitative evaluation of the degree of endometrial stromal cellular spacing was performed by counting stromal fibroblast nuclei/unit area (Histogram 2). The number of stromal cell count (mean±standard deviation) in the control group was 28±5.3, which was significantly higher than that in the PGE2 group (14.25±2.8, P=0.001) or in the EP4A group (13.75±3.0, P=0.001).
Alcian blue staining
This was manifested by weak staining as blue color in the control group (Fig. 4) that was mainly localized in the superficial portion of endometrial stroma beneath the luminal epithelium. In contrast, dense staining for Alcian blue stain was obtained in PGE2 and EP4A groups that occupied the major area of the stroma (Figs 5 and 6, respectively).
Collagen I immune staining
Dense staining for collagen I was observed in the control group (Figs 7 and 8). The staining was localized around the surface and glandular epithelia and at the interlacing bundles in the endometrial stroma. Weak immune staining was obtained in the PGE2 and EP4A groups compared with the control (Figs 9–11).
Hyaluronic acid immune staining
Positive staining for HA in the control group was regularly and diffusely distributed throughout the endometrial stroma with increased intensity particularly around the endometrial glands and blood vessels (Fig. 12). The PGE2 and EP4A groups demonstrated more intense staining (++ and ++, respectively) throughout the stroma compared with the control group (+) (Figs 13 and 14).
CD44 immune staining
Weak immune staining of CD44 was observed in the control group (Fig. 15). The staining was confined to the surface and glandular epithelium. No staining was noted in the endometrial stroma. Dense staining was seen in the PGE2 (Fig. 16) and EP4A (Fig. 17) groups compared with the control and with the same localization pattern.
Results of the previously mentioned three immune stainings assessed by the semiquantitative subjective scoring system are illustrated in Table 1.
Extensive studies are available in the literature with regard to the uterine endometrial changes during pregnancy, especially at the initial phases of the implantation process. Furthermore, many reports on the implication of PGE2 during the implantation window exist [17,18]. However, according to the available literature the impact of PGE2 or its receptor agonists on selected components of the endometrial extracellular matrix namely collagen I, GAGs, HA, and CD44 was not a subject of investigation. The implantation process involves edema of the endometrial stroma, disruption of the collagen fibrils, and alteration in GAGs [3–10]. This study showed that exogenous PGE2 and EP4A induced all these changes in the rat endometrium. Moreover, these PGE2 effects are likely mediated by the EP4 receptor at least in part.
In this study, HE staining showed that uterine endometrial stroma was edematous in the PGE2 and EP4A groups compared with the control. To verify this result, measurement of uterine weight in the three groups revealed a significant increase in the weight of PGE2 and EP4A groups compared with the control that probably indicates an increase in the intercellular substance. Furthermore, edema was quantitatively verified by stromal fibroblast cell count. In accordance with the results of this study, previous investigators demonstrated edematous changes in the rabbit uterine cervix after exogenous PGE2 treatment . In the cervix, edema is essential for cervical ripening before induction of labor. In the endometrium, edema serves many key roles. It provides tissue loosening required for implantation, supplies essential nourishment for the invading trophoblast [2,8], and may be the trigger for the decidual cell reaction. It is regarded as a key event for success of the implantation process. For this reason, edema is a constant finding at the initiation of the implantation process [3–10].
Numerous previous studies have demonstrated that the dense array of collagen fibrils in the endometrium is diminished with the decidual reaction [12,30,31], with advanced pregnancy, and with long-term castration [4,32]. Biochemical analysis of total collagen content revealed that the collagen concentration declines in decidual tissue [33,34]. Previously, immunohistochemical studies have shown a very low expression of collagen I in decidual tissue . Other studies detected decreased concentration of collagen I at implantation sites . Results in this study revealed a very low expression of collagen I immune staining after exogenous PGE2 and EP4A treatments and is in line with previous reports. Although the model used in this study was immature rat uterus, PGE2 and EP4A induced results similar to that found in pregnant uteri. In view of these results, it could be claimed that PGE2 (through EP4 receptor) plays a major role in collagen tissue disintegration in pregnant endometrium taking into consideration the fact that PGE2 has antifibrotic effects by inhibiting multiple fibroblast functions .
Selective degradation of the collagen fibrils during decidualization is associated with increased synthesis of highly hydrated space-filling molecules (GAGs) to stabilize the extracellular matrix. Recent investigations reported an increase in the synthesis of matrix GAGs by endometrial cells during the decidual response [8,37]. A unique and dramatic increase in the synthesis of HA on the day of implantation was noted in mouse [38,39]. It is assumed that increased HA biosynthesis during decidualization may promote embryo implantation and provide a more favorable environment for embryo development [38,40]. In this study, PGE2 and EP4A injections resulted in a significant increase in the total uterine matrix GAGs and HA as detected by the Alcian blue histochemistry and bHABP immune staining, respectively.
CD44 is a cell adhesion molecule that is expressed by many tissues [41–43]. It is believed to be involved in cell–cell and cell–matrix interactions. It is the receptor for HA, and thus it serves to maintain a hyaluronate-rich environment [15,44]. Apart from this function, the exact physiological roles of CD44 on the epithelium are not yet fully understood. Some researchers suggest that the pattern of CD44 expression in the uterine tissue plays a role in the adhesion phase of the implantation process . Extensive studies [10,45,46] reported a positive expression of the CD44 molecule at a time that corresponds to the implantation window during the secretory phase of the menstrual cycle. In this study, exogenous administration of both PGE2 and EP4A induced intense staining for the CD44 molecule at the surface and glandular epithelia. Moreover, treatment with PGE2 and EP4A resulted in dense immune staining for HA compared with the control group. Considering both results together, it could be concluded that PGE2 and EP4A resulted in accumulation of HA in the stroma and induced the expression of its receptor CD44 on the epithelia.
Collectively, it can be concluded that PGE2 is capable of creating an endometrial milieu that favors the success of the implantation process. EP4A injection demonstrated similar effects to PGE2 treatments. Thus, this study clarified that these PGE2 actions could be mediated through the EP4 receptor. Analysis using the synthesized agonists of PGE2 receptors will be able to provide direct evidence with regard to the mechanism of action of PGE2.
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