Skip to main content
Download PDF

Dysregulated erythropoietin-producing hepatocellular receptor A2 (EphA2) is involved in tubal pregnancy via regulating cell adhesion of the Fallopian tube epithelial cells

Reproductive Biology and Endocrinology volume 16, Article number: 84 (2018) Cite this article

Abstract

Background

Tyrosine kinase receptor erythropoietin-producing hepatocellular receptor A2 (EphA2) is abundant in the endometrium and plays a role in the establishment of eutopic implantation. A similar molecular mechanism may exist between uterine implantation and tubal implantation, therefore EphA2 involvement in tubal pregnancy is suspected. Due to the limited availability of human Fallopian tube specimens, EphA2 expression in human Fallopian tube epithelium remains largely unknown.

Methods

A total of 31 women with tubal pregnancy and 41 non-pregnant women with benign uterine diseases were enrolled in this study. Immunohistochemistry was used to investigate the expression pattern of EphA2 in the Fallopian tube epithelium of non-pregnant women (n = 29) and women with tubal pregnancy (n = 17). The changes of EphA2 and its activated form, phosphorylated-EphA2 (Pho-EphA2), in the Fallopian tube epithelium from non-pregnant women (n = 12) and women with tubal pregnancy (n = 14) were compared by quantitative RT-PCR and western blot assay.

Results

EphA2 was expressed throughout the Fallopian tube epithelium, including the isthmus, the ampulla and the infundibulum. EphA2 concentration remained unchanged throughout the whole menstrual cycle, irrespective of menstrual phases and tubal regions. EphA2 mRNA in the Fallopian tube epithelium did not differ between normal women and women with tubal pregnancy (P > 0.05). With respect to the protein level, a significantly higher ratio of EphA2 over Pho-EphA2 was shown in women with tubal pregnancy (P < 0.05).

Conclusions

EphA2 is widely expressed in human Fallopian tube epithelium in a temporospatial-independent manner. Dysregulated EphA2 and its phosphorylation-dependent regulatory mechanism may unexpectedly enhance the cell adhesion activity of the Fallopian tube epithelial cells, leading to a mis-contact between the Fallopian tube epithelium and the embryo.

Background

An ectopic pregnancy is a pathological event characterized by embryo implantation occurring outside the uterine cavity. Fallopian tube localization accounts for roughly 98% of all human ectopic pregnancies [1]. Tubal damage as a result of surgery or infection (especially Chlamydia trachomatis), smoking and in vitro fertilization are the dominant risk factors for tubal ectopic pregnancies [1]. However, tubal pregnancy is now thought to be a consequence of molecular dysregulation [1]. One major reason underlying this phenomenon is that adhesion molecules such as integrins and mucin 1, etc., which mediate blastocyst adhesion to the uterine wall, are also expressed in the Fallopian tube epithelium [2]. Unexpected molecular changes may afford an opportunity for the undue remain of embryo in the Fallopian tube [2, 3]. Cell adhesion molecules are abundant in human epithelial cells and crucial for intercellular adhesion. It is presumed that cell adhesion activity of the Fallopian tube epithelial cells is under the fine control of cell adhesion molecules and that changes in their concentrations can alter the cell adhesion of the Fallopian tube epithelium, leading to false recognition between mother and fetus [4,5,6]. Therefore, it is rational to propose that cell adhesion molecules expressed in the Fallopian tube epithelium may actively participate in the pathogenesis of tubal pregnancy.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) is a member of the largest known tyrosine kinase receptor family, which is well known to generate multiple cellular responses including cell adhesion and migration [7,8,9,10]. Activation of EphA2 is phosphorylation-dependent. Binding of EphA2 to its predominant EphrinA1 ligand enables its phosphorylation (Pho-EphA2) [11, 12]. This leads to the initiation of a downstream signaling cascade, which results in the internalization and degradation of Pho-EphA2 itself [11, 12]. It is found that EphA2 is highly localized in endometrial epithelial cells accompanied by its Ephrin ligand expressed on embryonic trophoblasts, and EphA2 is suggested to mediate the establishment of uterine implantation by regulating embryo-maternal contact [13, 14]. Furthermore, EphA2 can promote the invasion and proliferation of the human extravillous trophoblastic cells probably via regulating the ephrin-A1 ligand [15]. As a similar molecular mode may exist between uterine implantation and tubal implantation, it is reasonable to expect EphA2 involvement in tubal pregnancy. However, EphA2 expression in human Fallopian tube epithelium has not been fully documented and the role of EphA2 in tubal pregnancy remains unknown as yet.

We have previously demonstrated in vitro study that EphrinA1 could induce EphA2 activation in human fallopian tubal epithelial cells, accompanied by the up-regulation of Pho-EphA2 [16]. Moreover, we used fibronectin-coated culture plates to interact with the cultured Fallopian tube epithelial cells and found that EphA2 activation could attenuate its cell adhesion activity, which, when disrupted, may be associated with certain pathological events occurred in the Fallopian tube, such as tubal pregnancy [16]. However, the expression pattern of EphA2 in human Fallopian tube epithelium remains presently unknown. Therefore, the present study was undertaken to investigate the EphA2 expression in human Fallopian tube epithelium by immunohistochemical analysis. Considering the involvement of EphA2 activation in regulating cell adhesion activity of the Fallopian tube epithelial cells, the changes of EphA2 and its activated form, Pho-EphA2, in the Fallopian tube epithelium were further compared between non-pregnant women and women with tubal pregnancy using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot assay, aiming to elucidate the roles of EphA2 and its phosphorylation-dependent mechanism in the molecular pathogenesis of tubal pregnancy.

Methods

Specimen collection of human fallopian tubes

Thirty-one women who had been diagnosed as tubal pregnancy and undergone surgeries in the First Affiliated Hospital of Jinan University, PR China were collected, and 41 non-pregnant women who had undergone hysterectomies for benign uterine diseases were set as controls. Informed consent was attained from each patient and ethical approval for this study was obtained from the Local Research Ethics Committee. All the enrolled subjects should meet the following criteria: (1) a normal history of fertility and regular menses; (2) a definitive date of the last menstrual period; and (3) no use of exogenous hormonal drugs or intrauterine devices in the 6 months preceding the surgery. Women with tubal pregnancy who were determined to have morphological abnormalities in the Fallopian tubes or benign uterine diseases such as uterine leiomyoma, adenomyosis and endometriosis after surgical operations were excluded.

Among all the enrolled subjects, the Fallopian tube samples from 17 tubal pregnant women (mean age 27.7 ± 3.7 years; range 26–34 years; gestational weeks 8.5 ± 0.9) and 29 non-pregnant women (control) (mean age 37.6 ± 3.8 years; range 35–45 years) were paraffin embedded and used for immunohistochemistry. Each Fallopian tube sample with tubal pregnancy was further divided into 2 portions, implantation site (n = 17) and non-implantation site (n = 17), according to the distance apart from the gestational sac. The implantation site was defined as the circumference originating 5 mm from the gestation sac, while the non-implantation site was 10 mm outside the gestation sac [17]. The controls were further divided into 4 groups based on the corresponding endometrial morphology [18]: early-stage of the proliferative phase (days 1–5; n = 8), mid- and late-stages of the proliferative phase (days 7–14; n = 6), early-stage of the secretory phase (days 15–18; n = 7) and mid- and late-stages of the secretory phase (days 19–28; n = 8). All of the paraffin blocks were cut and prepared for immunohistochemical staining.

Fresh Fallopian tube samples obtained from the other 14 tubal pregnant women (mean age 28.9 ± 4.1 years; range from 28 to 37 years; gestational weeks 8.9 ± 0.9 weeks) and 12 non-pregnant women (mean age 40.3 ± 4.2 years; range from 35 to 46 years) were used for qRT-PCR and western blot analysis. Each Fallopian tube tissue with tubal pregnancy was similarly classified into implantation site and non-implantation site. Samples used for mRNA analysis were immersed into Trizol solution, and all fresh specimens were frozen at − 70 °C until analysis.

Immunohistochemistry

The paraffin-embedded materials were cut into 4-μm-thick sections. These sections were deparaffinized and boiled in citrate buffer solution for 15 min in an oven. After washing with phosphate-buffered saline (PBS), the sections were immersed into 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Afterwards, mouse anti-human EphA2 monoclonal antibody (1:200; ab118882; Abcam, Cambridge, UK) was applied overnight at 4 °C according to the EnVision™ system (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China). The sections were washed in PBS and incubated for 30 min with horse radish peroxidase (HRP)-conjugated goat anti-mouse IgG (BM2101; Dako Cytomatin, Glostrup, Denmark). Diaminobenzidine (DAB) substrate-chromogen system (Zhongshan Goldenbridge Biotechnology Co., Ltd., Beijing, China) was used as the color-developing substrate. The slides were applied in two 5-min incubations and then counter-stained with hematoxylin. Normal gastric mucosa with known presence of EphA2 served as a positive control. The primary antibody was replaced with non-immune mouse serum IgG to serve as a negative control. All slides were viewed under an E200 microscope (Nikon, Tokyo, Japan). Sections were quantified over ten randomly selected fields of view. Positive unit (PU) assessed by the QW550 image analysis system (Leica, Wetzlar, German) was used to describe the EphA2 expression [19, 20]. The formula was PU = 100 (Ga - Gb)/Gmax. Ga and Gb were the average gray scale of positive staining and background, respectively. Gmax was the maximum gray scale of image analytical system.

Quantitative RT-PCR

Total RNA was extracted from the fresh Fallopian tube samples using Trizol total RNA extraction reagent (Takara, Dalian, China). Agarose gel electrophoresis was used to ascertain the quality of the RNA products by the presence of 28S rRNA, 18S rRNA and 5S rRNA bands, with a 2:1 ratio of 28S rRNA to 18S rRNA. cDNA was reverse transcribed from the extracted RNA using RT kit (Takara, Dalian, China). The primers used for EphA2 were 5’-AAGACCCTGGCTGACTTT-3′ (forward) and 5’-GTTCACCTGGTCCTTGAGT-3′ (reverse), while the primers for 18sRNA were 5’-CCTGGATACCGCAGCTAGGA-3′ (forward) and 5’-CCTGGATACCGCAGCTAGGA-3′ (reverse). Real time RT-PCR was carried out using an ABI 7300 real-time PCR system (ABI, Carlsbad, USA) with 5 μl diluted cDNA and 0.5 μl primers. The programmed thermocycler included 40 cycles of predenaturation at 95 °C for 10 min, followed by denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and extension at 72 °C for 30 s. All reactions were performed in triplicate. Relative quantification of EphA2 mRNA was calculated using the comparative Ct method (ΔΔCt = ΔCt EphA2− ΔCt18sRNA).

Western blot

The total protein content extracted from the fresh Fallopian tube samples was resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h in 10% bovine serum albumin (BSA) (DingguoChangsheng Biotechnology Co., Ltd., Beijing, China) at room temperature, followed by incubation at 4 °C overnight with the primary antibodies, rabbit anti-human polyclonal antibodies against EphA2 and Pho-EphA2 (1:1000; 6997S and 6347S; Cell signaling technology Co., Ltd., Boston, USA) and mouse anti-human monoclonal antibody against glyceraldehyde phosphate dehydrogenase (GAPDH) (1:1000; sc-59,540; Santa Cruz, California, USA). After washing, the membranes were then incubated for 1 h with the appropriate HRP-conjugated secondary antibodies, goat anti-rabbit IgG (1:800; A0208; Beyotime Biotechnology Co., Ltd., Shanghai, China) or rabbit anti-mouse IgG (1:1000; D031402; Dako Cytomatin, Glostrup, Denmark). Protein bands were identified using ECL (Pierce, Rockford, USA). The bands were semi-quantified using Bio-Rad Quantity One software. EphA2 was normalized to GADPH, and Pho-EphA2 was normalized to EphA2.

Statistical analysis

All statistical analyses were carried out using SPSS V.14. (SPSS, Chicago, USA). Differences among groups were determined by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls (SNK) multiple-comparison test. Results were presented as mean ± standard deviation (SD). P values < 0.05 were considered statistically significant.

Results

EphA2 expressions in human fallopian tube epithelia of non-pregnant women

Immunohistochemistry confirmed that EphA2 was present throughout the Fallopian tube epithelium, embracing the isthmus, the ampulla and the infundibulum. EphA2 showed stronger staining on the apical membrane of both the ciliated cells and the secretory cells as well as the cilia. The cytoplasm of the ciliated cells and the secretory cells showed positive staining of EphA2, but the staining intensities in the cytoplasm were weaker than those on the apical membranes (Fig. 1a and b). No staining was detected in the interstitial cells. No significant differences of the staining intensities could be found among different sites of the Fallopian tube during the same menstrual phase or among different menstrual phases at the same tubal region (P > 0.05) (Table 1).

Fig. 1
figure 1

EphA2 immunostaining (brown) on the Fallopian tube epithelium during normal menstrual cycle. a EphA2 was expressed on the apical membrane (triangle) and in the cytoplasm (arrow) of the tubal epithelial cells, including the ciliated cells and the secretory cells, while the staining intensities in the cytoplasm were weaker than those on the apical membranes. There was no staining evident in the interstitial cells. b EphA2 was present in the cilia of the ciliated cells (arrow). c Stronger stains were shown on the apical membranes of the tubal epithelial cells. d Negative control

Full size image
Table 1 EhpA2 expressions in human Fallopian tube epithelia
Full size table

Changes of EphA2 and pho-EphA2 in human fallopian tube epithelia of women with tubal pregnancy

Immunohistochemical staining confirmed the presence of EphA2 in the Fallopian tube epithelia of women with tubal pregnancy, including the implantation site and the non-implantation site (Fig. 2a, b and c). The strongest staining of EphA2 was noted in the implantation group (10.16 ± 3.59), compared with that in the non-implantation group (8.03 ± 2.49) and the control group (6.20 ± 2.72) (P < 0.05). With regard to the latter two groups, the EphA2 staining intensities were similar (P > 0.05) (Fig. 2d).

Fig. 2
figure 2

EphA2 immunostaining (brown) on the Fallopian tube epithelium during tubal pregnancy. a EphA2 was present in the epithelial cells located in the implantation site of tubal pregnancy (arrow). b EphA2 was present in trophoblast cells found in the implantation site (arrow). c EphA2 was present in the non-implantation site of tubal pregnancy, and a stronger staining was evident on the cytomembrane (arrow) than that in the cytoplasm. d EphA2 expressions on the Fallopian tube epithelia with tubal pregnancy (n = 17) and the secretory Fallopian tube epithelia of normal controls (n = 15). EphA2 expression in the implantation group presented with the highest-level, compared with that in the non-implantation group and the control group (P < 0.05). EphA2 expressions in the latter two groups did not differ (P > 0.05)

Full size image

Western blot analysis revealed positive bands of EphA2 and Pho-EphA2 in both the implantation group and the non-implantation group (Fig. 3a). Similar to the results obtained from immunohistochemical assessment, the implantation group had a significantly increased level of EphA2 (2.08 ± 0.87) compared to that in the non-implantation group (1.41 ± 0.64) and the control group (1.44 ± 0.62) (P < 0.05). As for Pho-EphA2, a remarkably decreased level was seen in the implantation group (0.51 ± 0.25) compared to that in the non-implantation group (0.79 ± 0.37) and the control group (0.78 ± 0.35) (P < 0.05). However, both EphA2 and Pho-EphA2 levels did not differ between the non-implantation group and the control group (P > 0.05) (Fig. 3b).

Fig. 3
figure 3

Comparisons of EphA2 and Pho-EphA2 protein expression between the samples derived from the normal Fallopian tubes of the secretory phase (control) (n = 12) and the tubal pregnant tissues, which were further divided into the implantation group (n = 14) and the non-implantation group (n = 14). a Western blot showed positive bands of EphA2 and Pho-EphA2 in both the implantation and non-implantation groups of tubal pregnancy. b The highest EphA2 accompanied by the lowest Pho-EphA2 was detected in the implantation group, compared with that in the non-implantation group and the control group (P < 0.05). EphA2 and Pho-EphA2 protein expressions in samples of the latter two groups were similar (P > 0.05)

Full size image

EphA2 mRNA in human fallopian tube epithelia between non-pregnant women and women with tubal pregnancy

The EphA2 mRNA levels were similar among the implantation site, the non-implantation site and the Fallopian tube from non-pregnant women (control), with the corresponding values referring to 1.94 ± 0.51, 1.85 ± 0.74 and 2.02 ± 0.55, respectively (P > 0.05) (Fig. 4).

Fig. 4
figure 4

EphA2 mRNA levels did not differ among the control group (n = 12), the implantation group (n = 14) and the non-implantation group (n = 14) (P > 0.05)

Full size image

Discussion

To the best of our knowledge, this is the first study to compare the expressions of EphA2 in human Fallopian tube epithelia between non-pregnant women and women with tubal pregnancy. Acted like the endometrium, the morphology of the Fallopian tube epithelium could similarly respond to a menstrual cycle [21, 22]. Therefore, in the present study, the normal Fallopian tube samples were categorized according to the corresponding endometrial morphology, and EphA2 was revealed to widely express in human Fallopian tube epithelium in a temporospatial-independent manner. An increased EphA2 accompanied by a decreased Pho-EphA2 was noted in the Fallopian tube epithelium with tubal pregnancy. EphA2 activation is autophosphorylation-dependent and the elevated Pho-EphA2, induced by EphA2 activation, could attenuate the cell adhesion activity of human Fallopian tube epithelial cells [16]. Combined with the present data, it is speculated that the aberrant EphA2 in the Fallopian tube epithelium with tubal pregnancy, as indicated by a decreased ratio of Pho-EphA2 over EphA2, may unexpectedly enhance cell adhesion activity the of Fallopian tube epithelial cells, leading to a mis-contact between the Fallopian tube epithelium and the embryo.

Investigation of pathological events occurring within the Fallopian tube necessitates considerations of the tubal microenvironment, of which the homeostasis is largely regulated by the molecules expressed on the Fallopian tube epithelium. Tubal pregnancy is currently accepted to be a direct consequence of molecular causes, considering that molecules capable of mediating cell adhesion have been confirmed to play a critical role in the pathogenesis of tubal pregnancy [23]. Eph receptor tyrosine kinases as well as their ligands, ephrins, are important regulators in cell-to-cell interaction and have been proven to play crucial roles in cell migration and adhesion during embryonic development in mammals [24]. EphA2 is abundant in human endometrial epithelial cells and its ligand ephrin is expressed in the blastocysts. A previous study revealed that the Eph-ephrin A system could regulate the initial embryo-maternal contact during the cross-talk period that preceded embryo implantation in the endometrium [14]. Similar regulatory mechanism may exist in the process of tubal implantation. However, evidence pertaining to the role of EphA2 in tubal pregnancy is scarce. The present study revealed that EphA2 was presented in both the ciliated and secretory cells of the Fallopian tube epithelia, and a temporospatial-independent pattern of EphA2 expression was noted. The levels of EphA2 in the Fallopian tube epithelia remained unchanged throughout the whole menstrual cycle, irrespective of menstrual phase or Fallopian tube region. The morphology and physiology of the Fallopian tube epithelium, including the ciliated and secretory cells, can be regulated by sex hormones fluctuating with the menstrual cycle [21]. Such a temporospatial-independent pattern may, in a sense, imply that sex hormones have no impact on EphA2 expression in the Fallopian tube epithelium.

It is well-recognized that overexpression of EphA2 is merely presented in malignant cells, but in non-transformed epithelial cells, EphA2 displays a relatively low level of expression [25,26,27]. Similar to this pattern, EphA2 was noted to be less abundant in the normal Fallopian tube epithelial cells in contrast to a significant increase in those from women with tubal pregnancy. One possible explanation for this phenomenon may be that EphA2 and its regulatory mechanism are initiated improperly under certain pathological conditions. Moreover, EphA2 is reported to not only function as a marker in transformed cells, but also possess the property to aggravate the malignant progression. Overexpression of EphA2 in non-transformed mammary epithelial cells is sufficient to promote a malignant phenotype [27]. Thus, we tended to speculate that the overexpression of EphA2 presented in the Fallopian tube epithelium may represent its active participation in tubal pregnancy via increasing the cell adhesion activity of the Fallopian tube epithelial cells but not only a simple result caused by tubal implantation.

An increased level of EphA2 was shown in the implantation group compared with that in the non-implantation group and the control group by western blot. However, it seemed a paradox that we failed to find any discrepancy in EphA2 mRNA in the Fallopian tube samples among these three groups. Previous studies concerning the linkage between EphA2 and malignant tumors indicated that an elevated EphA2 found in malignant tumors was a result of deficient phosphorylation of EphA2 itself, leading to the consequent blockage of EphA2 degradation. In other words, insufficient Pho-EphA2 could accordingly give rise to an excessive storage of EphA2 [28, 29]. This conclusion may shed some light on the present data. The presence of increased EphA2 was accompanied by a decrease in Pho-EphA2, implying that the homeostasis of EphA2 in the Fallopian tube epithelium was hampered during tubal pregnancy, and the increased level of EphA2 may occur on the basis of insufficient phosphorylation of EphA2 itself. The activation of EphA2 is coupled with its autophosphorylation, characterized by an elevated ratio of Pho-EphA2 over EphA2, which could attenuate the cell adhesion activity of the Fallopian tube epithelial cells [16]. In the present study, a decreased ratio of Pho-EphA2 over EphA2 was detected in the implantation site of the Fallopian tube epithelium. We hypothesized that this may disclose an inactivated state of EphA2, which would on the contrary unexpectedly enhance the cell adhesion activity of the Fallopian tube epithelium. Thereafter, a false recognition between fetus and mother may be evoked to take place within the specific site of the Fallopian tube epithelium.

Ciliary motion is closely associated with female fecundity by regulating the processes of ovum pickup and transport [30]. Altered activity of cilia was reported to play a pivotal role in the pathogenesis of tubal pregnancy, based on the fact that women presenting with decreased cilia consequently suffered from tubal pregnancy [31]. EphA2/Ephrin-A1 signaling could attenuate cell adhesion activity of the Fallopian tube epithelial cells [16]. EphA2 was shown in the cilia of the tubal ciliated cells, suggesting a possibility that EphA2 may have some implications in the modulation of ciliary activity by regulating cell adhesion. However, the precise mechanisms about how the inactivated EphA2, characterized in the present study as an increased ratio of EphA2 over Pho-EphA2, influenced the ciliary motion still need to be further elucidated.

There are still certain limitations in the present study we need to take into consideration. Firstly, there were a limited number of samples included in this study which may weaken the strength of our evidence. Secondly, there were some age differences between non-pregnant women and women with tubal pregnancy since it is difficult to obtain Fallopian tube specimens from normal young non-pregnant women for ethical reasons. Lastly, Fallopian tube samples from women with tubal pregnancy can only be collected after an implantation event has occurred, making it difficult to directly identify the causal linkage between EphA2 and tubal pregnancy.

Many efforts have been made to clarify the mechanisms responsible for EphA2 activation during pathological events, yet there is still largely unexplored [32,33,34]. EphA2 activation can suppress tumor cell growth and hence EphA2 has been established as a therapeutic target for malignant tumors by hindering the progression of tumor invasion [35]. Known to be one of the key molecules responsible for regulating cell adhesion activity, it is plausible to assume that EphA2 activation and its downstream signaling could also be established as a molecular target in medical therapy for tubal pregnancy by decreasing cell adhesion activity of the Fallopian tube epithelial cells to avoid the mis-recognition at the maternal-fetal interface.

Conclusions

In conclusion, we have demonstrated that EphA2 is expressed in human Fallopian tube epithelium in a temporospatial-independent manner. EphA2 and its phosphorylation-dependent regulatory mechanism seem to be activated only under pathological conditions. The presence of a reduced Pho-EphA2 accompanied by an elevated EphA2, a state that may be recognized as EphA2 inactivation, maybe involved in the molecular pathogenesis of tubal pregnancy through up-regulating cell adhesion activity of the Fallopian tube epithelial cells. A better understanding of EphA2 and its signal pathways is of significance to illustrate the molecular pathogenesis of tubal pregnancy. Further investigations with larger samples are necessary to complement our present study.

Abbreviations

BSA:

Bovine serum albumin

DAB:

Diaminobenzidine

EphA2:

Erythropoietin-producing hepatocellular receptor A2

GAPDH:

Glyceraldehyde phosphate dehydrogenase

HRP:

Horse radish peroxidase

PBS:

Phosphate-buffered saline

Pho-EphA2:

Phosphorylated EphA2

PU:

Positive unit

qRT-PCR:

Quantitative reverse transcription-polymerase chain reaction

SDS-PAGE:

Sodium dodecylsulfate-polyacrylamide gel electrophoresis

References

  1. Shaw JL, Dey SK, Critchley HO, Horne AW. Current knowledge of the aetiology of human tubal ectopic pregnancy. Hum Reprod Update. 2010;16(4):432–44.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Brito LR, Guedes Neto EP, Furich DG, Savaris RF. MUC1 (VPM654 and EPR1023) expression in mucosa of fallopian tubes with ectopic pregnancy is altered. Appl Immunohistochem Mol Morphol. 2016;24(8):569–74.

    Article  PubMed  CAS  Google Scholar 

  3. Zhang JR, Li SD, Wu XP. Eutopic or ectopic pregnancy: a competition between signals derived from the endometrium and the fallopian tube for blastocyst implantation. Placenta. 2009;30(10):835–9.

    Article  CAS  Google Scholar 

  4. Bowen JA, Hunt JS. The role of integrins in reproduction. Proc Soc Exp Biol Med. 2000;223(4):331–43.

    Article  PubMed  CAS  Google Scholar 

  5. Makrigiannakis A, Karamouti M, Petsas G, Makris N, Nikas G, Antsaklis A. The expression of receptivity markers in the fallopian tube epithelium. Histochem Cell Biol. 2009;132(2):159–67.

    Article  PubMed  CAS  Google Scholar 

  6. Utreras E, Ossandon P, Acuna-Castillo C, Varela-Nallar L, Muller C, Arraztoa JA, Cardenas H, Imarai M. Expression of intercellular adhesion molecule 1 (ICAM-1) on the human oviductal epithelium and mediation of lymphoid cell adherence. J Reprod Fertil. 2000;120(1):115–23.

    Article  PubMed  CAS  Google Scholar 

  7. Carter N, Nakamoto T, Hirai H, Hunter T. EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas). Nat Cell Biol. 2002;4(8):565–73.

    Article  PubMed  CAS  Google Scholar 

  8. Davy A, Aubin J, Soriano P. Ephrin-B1 forward and reverse signaling are required during mouse development. Genes Dev. 2004;18(5):572–83.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Zhao C, Irie N, Takada Y, Shimoda K, Miyamoto T, Nishiwaki T, Suda T, Matsuo K. Bidirectional ephrinB2-EphB4 signaling controls bone homeostasis. Cell Metab. 2006;4(2):111–21.

    Article  PubMed  CAS  Google Scholar 

  10. Lim BK, Matsuda N, Poo MM. Ephrin-B reverse signaling promotes structural and functional synaptic maturation in vivo. Nat Neurosci. 2008;11(2):160–9.

    Article  PubMed  CAS  Google Scholar 

  11. Nasreen N, Mohammed KA, Lai Y, Antony VB. Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM). Cancer Lett. 2007;258(2):215–22.

    Article  PubMed  CAS  Google Scholar 

  12. Lema Tome CM, Palma E, Ferluga S, Lowther WT, Hantgan R, Wykosky J, Debinski W. Structural and functional characterization of monomeric EphrinA1 binding site to EphA2 receptor. J Biol Chem. 2012;287(17):14012–22.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Goldman-Wohl D, Greenfield C, Haimov-Kochman R, Ariel I, Anteby EY, Hochner-Celnikier D, Farhat M, Yagel S. Eph and ephrin expression in normal placental development and preeclampsia. Placenta. 2004;25(7):623–30.

    Article  PubMed  CAS  Google Scholar 

  14. Fujii H, Tatsumi K, Kosaka K, Yoshioka S, Fujiwara H, Fujii S. Eph-ephrin a system regulates murine blastocyst attachment and spreading. Dev Dyn. 2006;235(12):3250–8.

    Article  PubMed  CAS  Google Scholar 

  15. Yang Y, Min J. Effect of ephrin-A1/EphA2 on invasion of trophoblastic cells. J Huazhong Univ Sci Technolog Med Sci. 2011;31(6):824–7.

    Article  PubMed  CAS  Google Scholar 

  16. Yang XY, Zhu WJ, Jiang H. Activation of erythropoietin-producing hepatocellular receptor A2 attenuates cell adhesion of human fallopian tube epithelial cells via focal adhesion kinase dephosphorylation. Mol Cell Biochem. 2012;361(1–2):259–65.

    Article  PubMed  CAS  Google Scholar 

  17. Lam PM, Briton-Jones C, Cheung CK, Leung SW, Cheung LP, Haines C. Increased messenger RNA expression of vascular endothelial growth factor and its receptors in the implantation site of the human oviduct with ectopic gestation. Fertil Steril. 2004;82(3):686–90.

    Article  PubMed  CAS  Google Scholar 

  18. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol. 1975;122(2):262–3.

    Article  PubMed  CAS  Google Scholar 

  19. Murray TJ, Fowler PA, Abramovich DR, Haites N, Lea RG. Human fetal testis: second trimester proliferative and steroidogenic capacities. J Clin Endocrinol Metab. 2000;85(12):4812–7.

    PubMed  CAS  Google Scholar 

  20. Harrison JL, Adam CL, Brown YA, Wallace JM, Aitken RP, Lea RG, Miller DW. An immunohistochemical study of the localization and developmental expression of ghrelin and its functional receptor in the ovine placenta. Reprod Biol Endocrinol. 2007;5:25.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Zhu J, Xu Y, Rashedi AS, Pavone ME, Kim JJ, Woodruff TK, Burdette JE. Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle. Mol Hum Reprod. 2016;22(11):756–67.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Bauersachs S, Rehfeld S, Ulbrich SE, Mallok S, Prelle K, Wenigerkind H, Einspanier R, Blum H, Wolf E. Monitoring gene expression changes in bovine oviduct epithelial cells during the oestrous cycle. J Mol Endocrinol. 2004;32(2):449–66.

    Article  PubMed  CAS  Google Scholar 

  23. Kemp B, Kertschanska S, Kadyrov M, Rath W, Kaufmann P, Huppertz B. Invasive depth of extravillous trophoblast correlates with cellular phenotype: a comparison of intra- and extrauterine implantation sites. Histochem Cell Biol. 2002;117(5):401–14.

    Article  PubMed  CAS  Google Scholar 

  24. Fujiwara H, Yoshioka S, Tatsumi K, Kosaka K, Satoh Y, Nishioka Y, Egawa M, Higuchi T, Fujii S. Human endometrial epithelial cells express ephrin A1: possible interaction between human blastocysts and endometrium via Eph-ephrin system. J Clin Endocrinol Metab. 2002;87(12):5801–7.

    Article  PubMed  CAS  Google Scholar 

  25. Coffman KT, Hu M, Carles-Kinch K, Tice D, Donacki N, Munyon K, Kifle G, Woods R, Langermann S, Kiener PA, Kinch MS. Differential EphA2 epitope display on normal versus malignant cells. Cancer Res. 2003;63(22):7907–12.

    PubMed  CAS  Google Scholar 

  26. Kinch MS, Carles-Kinch K. Overexpression and functional alterations of the EphA2 tyrosine kinase in cancer. Clin Exp Metastasis. 2003;20(1):59–68.

    Article  PubMed  CAS  Google Scholar 

  27. Walker-Daniels J, Hess AR, Hendrix MJC, Kinch MS. Differential regulation of EphA2 in normal and malignant cells. Am J Pathol. 2003;162(4):1037–42.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Miao H, Burnett E, Kinch M, Simon E, Wang B. Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation. Nat Cell Biol. 2000;2(2):62–9.

    Article  PubMed  CAS  Google Scholar 

  29. Brantleysieders DM, Zhuang G, Hicks D, Fang WB, Hwang Y, Cates JM, Coffman K, Jackson D, Bruckheimer E, Muraokacook RS. The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling. J Clin Invest. 2008;118(1):64–78.

    Article  CAS  Google Scholar 

  30. Noreikat K, Wolff M, Kummer W, Kolle S. Ciliary activity in the oviduct of cycling, pregnant, and muscarinic receptor knockout mice. Biol Reprod. 2012;86(4):120.

    Article  PubMed  CAS  Google Scholar 

  31. Lyons RA, Saridogan E, Djahanbakhch O. The reproductive significance of human fallopian tube cilia. Hum Reprod Update. 2006;12(4):363–72.

    Article  PubMed  CAS  Google Scholar 

  32. Dohn M, Jiang J, Chen X. Receptor tyrosine kinase EphA2 is regulated by p53-family proteins and induces apoptosis. Oncogene. 2001;20(45):6503–15.

    Article  PubMed  CAS  Google Scholar 

  33. Kikawa KD, Vidale DR, Van Etten RL, Kinch MS. Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation. J Biol Chem. 2002;277(42):39274–9.

    Article  PubMed  CAS  Google Scholar 

  34. Saito T, Masuda N, Miyazaki T, Kanoh K, Suzuki H, Shimura T, Asao T, Kuwano H. Expression of EphA2 and E-cadherin in colorectal cancer: correlation with cancer metastasis. Oncol Rep. 2004;11(3):605–11.

    PubMed  CAS  Google Scholar 

  35. Noblitt LW, Bangari DS, Shukla S, Mohammed S, Mittal SK. Immunocompetent mouse model of breast cancer for preclinical testing of EphA2-targeted therapy. Cancer Gene Ther. 2005;12(1):46–53.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. Ling-Juan Wu and Dr. Richard Daniel, CBCB, Newcastle University, UK, for critical reading of this manuscript.

Availability of data and materials

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

Author information

Authors and Affiliations

  1. Department of Reproductive Endocrinology, Longgang District Maternal and Child Healthcare Hospital, 6# Ailong Road, Longgang Central District, Shenzhen City, 518172, People’s Republic of China

    Huan Jiang

  2. Institute of Reproductive Immunology, College of Life Science and Technology, Jinan University, 601# Huangpu Da Dao Xi, Guangzhou City, 510632, People’s Republic of China

    Xiao-Yi Yang & Wei-Jie Zhu

Authors
  1. Huan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao-Yi Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei-Jie Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HJ and XYY contributed equally to this study. HJ, XYY and WJZ contributed to the conception and design of this study. XYY acquired the data. HJ and XYY performed the data analysis. All authors assisted in interpretation of data. HJ drafted the manuscript and WJZ revised it critically. All authors read and gave final approval of the manuscript.

Corresponding author

Correspondence to Wei-Jie Zhu.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Local Research Ethics Committee, and informed consent was attained from each patient.

Consent for publication

N/A

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jiang, H., Yang, XY. & Zhu, WJ. Dysregulated erythropoietin-producing hepatocellular receptor A2 (EphA2) is involved in tubal pregnancy via regulating cell adhesion of the Fallopian tube epithelial cells. Reprod Biol Endocrinol 16, 84 (2018). https://doi.org/10.1186/s12958-018-0403-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12958-018-0403-5

Keywords

Reproductive Biology and Endocrinology

ISSN: 1477-7827

Contact us