Distribution of estrogen receptor in the rabbit cervix during pregnancy with special reference to stromal elements: an immunohistochemical study

The estrogen plays a critical role during pregnancy through their receptors. Although the rabbit is one of the most important lab animal estrogen receptor alpha (ERA) localization on basic cells, newly discovered cells including telocyte and neuroendocrine cells, vascular compartments and interstitium during pregnancy not been described. At 0 day pregnancy, the most prominent immunoreactivity was moderate to ERA and observed on the ciliated cells, secretory cells, blood plasma, and interstitium. The smooth muscles and the endothelial cells showed mild immunoreactivity to ERA. Lymphocytes only exhibited strong immunoreactivity to ERA. At 7 days pregnancy moderate immunoreactivity to ERA observed on ciliated cells, secretory cells, smooth muscles, interstitium, and lymphocytes. Strong immunoreactivity to ERA detected on endothelial cells and blood plasma. At 14 days of pregnancy, the most prominent immunoreactivity was strong and detected on ciliated cells, smooth muscles, lymphocytes, and interstitium. Moderate immunoreactivity detected on endothelial cells and blood plasma. Secretory cells only exhibited mild immunoreactivity to ERA. At 21 days of pregnancy, the immunoreactivity to ERA ranged between mild on ciliated cells, smooth muscles, blood plasma and interstitium and negative on secretory cells, endothelial cells and lymphocytes. Our results indicated that the frequency and intensity of ERA immunostaining in the rabbit cervix varied on different structural compartments of the cervix during different pregnancy stages.


Material and methods
The current research was performed according to the Egyptian laws and guidelines of University for animal care. Faculty of Veterinary Medicine National Ethical Committee, Assiut University, Egypt, has authorized all the steps in the present work.
Sample collection. The study was approved by the Committee of Use and Care of experimental animals of Faculty of Veterinary Medicine, Assiut University, Egypt. The cervix was collected from the female genitalia of 12 healthy New Zealand white rabbits does (3 for each of the following pregnancy days) at 0, 7, 14 and 21 days of pregnancy. The day of mating is considered the zero day of pregnancy and then we collected the cervix of the pregnant does after 7 days, 14 and 21 days. we detected the pregnant does by palpation method and ultrasonography. The rabbit cervix was dissected immediately after slaughtering and fixed in Bouin's solution.
immunohistochemistry. Paraffin sections of (5 µm) were dewaxed by xylene, rehydrated by ascending grades of alcohol and rinsed by PBS pH 7.4 (3 times for 5 min). Endogenous peroxidase was suppressed by using hydrogen Peroxide block at room temperature. The sections were thoroughly washed by running tap water for an additional 10 min. To enhance antigen retrieval, the slides were treated with 10 mm sodium citrate buffer (pH 6.0) at temperature reached 95-98 in a water bath for 20 min. The sections were cooled for 20 min at room temperature and subsequently were washed in PBS (pH 7.4, 3 times for 5 min). Block non-specific background staining was performed by using Ultra V block, for 5 min at room temperature. Ultra V block application did not exceed 10 min to avoid staining artifact). The primary antibody was applied to the sections overnight at 4 °C. The used primary antibody was a mouse anti-rabbit antibody against Estrogen receptor alpha (ERA) Rabbit (PolyClonal; Cat. #RM-9101-S0 From ThermoFisher Scientific/Lab Vision) at dilution (1:200) in the PBS for one hour at room temperature. Sections were washed using PBS (at pH 7.4, 3 times for 5 min).
The biotinylated secondary antibody was applied for 10 min at room temperature. The Biotinylated secondary antibody was Biotinylated goat Anti-Polyvalent, Anti-mouse igg + Anti -Rabbit igg, Thermo Fisher Scientific, The UK. Lab Vision Corporation, ready to use. Sectioned were washed by PBS (pH 7.4, 3 times for 5 min) and subsequently incubated with streptavidin-peroxidase complex. Thermo Fisher Scientific, UK. LabVision corporation; USA) for10 min at room temperature. Visualization of the bound antibodies was performed using 1 drop of DAB plus chromogen to 2 mL of DAB plus substrate. The mixture was applied and incubated at room temperature for 5 min. The incubation processes were carried out in a humid chamber. Harris hematoxylin was used as counters stained for 30 s. The sections were dehydrated using ethanol and isopropanol I and II, cleared in xylene and covered by DPX 31,32 .
The expression of ERA in the cervix was examined microscopically using OLYMPUS BX51 microscope and the images were taken using OLYMPUS DP72 camera adapted to the microscope. By assessing the intensity of the immunostaining, the staining of the nucleus and/or cytoplasm by the following amount and color of immunostaining: strong (dark brown to black), moderate (brown), mild (light brown) and negative immunostaining (no immunoreactivity) 33 .
Percentage of immunohistochemical positive cells for ERA at different stages of pregnancy were measured using Image-J software. The measurements were carried on 9 section per animal (from each section 3 different areas were measured). Collected data were subjected to the Analysis of Variance (ANOVA) using Statistical Analysis System (SAS). Data were depicted as means ± SEM to show mean data deviation.

Results
Immunohistochemical reactivity described on cervical lining epithelium, intraepithelial lymphocyte, neuroendocrine cells, smooth muscle fibers, blood vessels, lymph vessels, interstitium and different kinds of connective tissue elements including fibroblast, lymphocytes, and telocyctes.
At 0 day of pregnancy. At 0 day of pregnancy, immunohistochemical localization of ERA in the rabbit cervix showed moderate cytoplasmic and nuclear immunostaining in both ciliated and secretory cells distributed randomly within the cervical lining epithelium. However, Most of the cervical lining epithelium showed negative immunostaining to ERA (Fig. 1A,B). The intraepithelial lymphocytes showed mild, moderate to strong cytoplasmic immunostaining to ERA (Fig. 1A,C,D). In addition, the subepithelial telocytes had moderate cytoplasmic and negative nuclear ERA immunoreactivity (Fig. 1E). Also, different kinds of cells within the lamina propria including fibroblasts and lymphocytes exhibited mild cytoplasmic and negative nuclear ERA immunoreactivity (Fig. 1F). Mild immunoreactivity of ERA was expressed in the cytoplasm and nucleus of some of the smooth muscle fibers of the cervix (Fig. 1G). Moreover, the blood plasma, red blood cells and cytoplasm of leukocyte within the arteries and veins demonstrated moderate immunoreactivity to ERA. Lymph within lymphatic vessels also showed strong immunoreactivity to ERA. Also, some of the endothelial cells showed mild ERA immunostaining and the stroma surrounding the blood vessels exhibited mild and strong ERA immunostaining (Fig. 1H). Serosal interstitum and mesothelium showed moderate and strong ERA immunostaining (Fig. 1I). www.nature.com/scientificreports/ At 7 days of pregnancy. At 7 days of pregnancy, moderate cytoplasmic and moderate nuclear immunostaining to ERA in secretory cells. However, ciliated cells exhibited moderate cytoplasmic and negative nuclear immunostaining. On the other hand, Most of the cervical lining epithelium showed negative immunostaining to ERA and positive cells distributed randomly within the cervical lining epithelium ( Fig. 2A,B). The intraepithelial lymphocytes showed moderate cytoplasmic ERA immunostaining. Intraepithelial neuroendocrine cells expressed mild to strong cytoplasmic and nuclear immunostaining to ERA and distributed singly or in groups (Fig. 2C). In addition, the subepithelial telocytes showed moderate cytoplasmic and mild nuclear immunostaining to ERA (Fig. 2D).
Our results indicated that mild cytoplasmic and moderate to strong nuclear immunoreactivity of ERA was expressed in some of the smooth muscle fibers of the cervix (Fig. 3A). Moreover, the endothelium of the blood vessels showed strong immunoreactivity to ERA and the blood plasma within the arteries and veins demonstrated strong immunoreactivity to ERA (Figs. 2D and 3B). Serosal interstitium and mesothelium showed strong ERA immunostaining (Fig. 3C,D). At 14 days of pregnancy. At 14 days of pregnancy, the rabbit cervix expressed strong immunostaining to ERA as a whole. Regarding the cervical lining epithelium, we observed that secretory cells demonstrated strong cytoplasmic and nuclear immunostaining to ERA but ciliated cells showed mild cytoplasmic and nuclear immunostaining to ERA (Fig. 4A). The intraepithelial lymphocytes indicated moderate to strong cytoplasmic and mild to moderate nuclear immunostaining to ERA (Fig. 4B). Telocytes in lamina propria showed a strong www.nature.com/scientificreports/ cytoplasmic and moderate nuclear reaction to ERA (Fig. 4C). Strong cytoplasmic and strong nuclear immunoreactivity of ERA was expressed in many of smooth muscle fibers of the cervix (Fig. 4D). Also, the endothelial cells of the blood vessels showed moderate ERA immunostaining (Fig. 4E). Serosal mesothelium showed strong ERA immunostaining (Fig. 4F). Also, blood plasma and interstitium showed moderate and strong immunoreactivity to ERA respectively (Fig. 4G).
At 21 days of pregnancy. At 21 days of pregnancy, few ciliated cells and few smooth muscle fibers showed mild immunostaining to ERA. In addition, the blood plasma showed mild immunostaining to ERA. Interstitium exhibited mild immunostaining to ERA (Fig. 5A-D).
Overall, assessment of the immunoreactions indicated that the cervix of pregnant rabbits had different immunostaining to ERA at 0, 7, 14 and 21 days of pregnancy. The staining intensity was mild at 0 day, moderate at 7 days, strong at 14 days and mild at 21 days of pregnancy (Fig. 6A-D). Also, the summary of ERA immunoreaction occurred on different cervical structural components summarized in Fig. 7.
Percentage of ERA positive cells at different stages of pregnancy were demonstrated on Table 1.

Discussion
Throughout pregnancy, the cervix has a load-bearing function to withstand the pressure from the growing fetus and not dilate during pregnancy 34 . Cervical ripening is a complex process occurring gradually to facilitate the expulsion of the fetus 35 . Several changes observed during these processes including cellular and extracellular matrix elements 36 . Moreover, cervical remodeling occurred through the modulating effects of estrogen receptors 37 . Also, at late pregnancy stages the cervical levels of the estrogen receptor are low 21 . www.nature.com/scientificreports/ The present study demonstrated the presence of ERA in the rabbit cervix during pregnancy. Estrogen action is mediated primarily by binding to specific intracellular receptors in target cells 38 . Our study provides new information regarding the expression and localization of ERA in the normal rabbit cervix during different pregnancy stages. We determined that in the rabbit cervix, ERA displayed differences among cell types at 0, 7, 14 and 21 days of pregnancy. Based on the density of ERA, these may provide a favorable environment throughout pregnancy. Moreover, in the pregnant sheep and in women estrogen administration promotes the softening of the cervix under the experimental conditions 39 . However, the cervical softening in a pig not promoted by estrogen-only 40 .
The cervical lining epithelium of the rabbit consisted mainly of ciliated and secretory cells 22 . Our data revealed that ciliated cells exhibiting mild to strong immune staining to ERA and secretory cells showed negative to moderate immunostaining to ERA during different pregnancy stages. Estrogen was very important for the function of the ciliated cell during transport through the uterine tube and induce important secretory products of secretory cells 41 . The endocrine role of cervical lining epithelium preserves cervical function during pregnancy 42 .
Neuroendocrine cells observed on different parts of the female reproductive tract in non-pregnant conditions 29,43 . Our result indicated that neuroendocrine cells take an intraepithelial position and showed different immunoreactivity to ER.
Our data revealed than intraepithelial and subepithelial lymphocyte of the cervix during pregnancy showed from negative to strong positive immunoreactivity to ER. Estrogen plays a key role in the regulation of immune cells of the female reproductive tract 44 . Intraepithelial lymphocyte expresses positive immunoreactivity to ER in the uterine tube. Also, described previously in the management of local immune tolerance during the movement of sperm on the uterine tube 45 .
Our result indicated that stroma cells as fibroblasts showed immunoreactivity to ERA. Also, fibroblasts act as hormone receptors and through paracrine activity effect on epithelial cells 46 . Moreover, steroid hormones play an important role in the proliferation and differentiation of stromal cells and the maintenance of pregnancy. There www.nature.com/scientificreports/ were paracrine dialog between the epithelial lining of uterus and stroma cells controlled by steroid hormone receptors in these cells to regulate its function during pregnancy 47 . Telocyte was newly discovered interstitial cell with paracrine activity and play a role in tissue repair and regeneration 33,48,49 . Also, it exhibited immunoreactivity to different steroid hormones 31,50,51 . Telocyte previously observed on the female reproductive tract 32,52-54 . Our results revealed that telocyte express moderate to strong immunoreactivity to ER during different stages of pregnancy. In addition, telocyte act as a steroid sensor on the female reproductive tract 55,56 .
The action of estrogen on smooth muscle was important for a successful pregnancy. Our data revealed that ERA expression on the smooth muscle fibers of the cervix increase gradually to reach the peak at mid-pregnancy then decreases at late pregnancy. These may be strongly related to the function of the cervix to be strongly closed during pregnancy and began to dilate at end of pregnancy which opposite which happened on the myometrium. The low expression estrogen receptor at early pregnancy help in myometrial quiescence and the increase of estrogen receptor expression until term pregnancy induce myometrial contraction during parturition these on the help of other hormones action 57 .
We described the expression of ERA at blood and lymph vessels during pregnancy which ranges between weak to strong. In addition, the role of estrogen described before on the blood vessels during pregnancy including growth and dilation to improve blood flow 58 . Also, in our results red and white blood cells showed www.nature.com/scientificreports/ immunoreactivity to ERA. Moreover, function of white blood cells was controlled by sex steroid hormones 59 . The expression of estrogen receptor on leukocytes endothelial cells of the cervical blood vessels were important in the regulation of white blood cells function in the induction of certain enzymes responsible for cervical remolding during pregnancy 60 . Also, the expression of ERA in RBCs observed by their effect on the function of RBCs as scavengers to reactive oxygen and nitrogen species 61 .
In the last few years, several studies began to focus on interstitium as fluid-filled spaces described on the gastrointestinal tract, dermis, and urinary bladder. Also, interstitium may be important on the mechanical structure of these organs and play a role in fibrosis and cancer metastasis 62 . In our result, the interstitium contains interstitial fluid which showed moderate immunostaining at early pregnancy, strong immunostaining at midpregnancy and mild immunostaining at late pregnancy for ERA these indicate the strong correlation between the interstitium and the cervical remodeling and cervical ripening. In addition, our data revealed that various fluid present on the cervix exhibiting immunoreactivity to ERA with different intensity throughout pregnancy including, blood plasma and lymph.  www.nature.com/scientificreports/

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.