Uterine NDRG2 expression is increased at implantation sites during early pregnancy in mice, and its down-regulation inhibits decidualization of mouse endometrial stromal cells

Background N-myc down-regulated gene 2 (NDRG2) is a tumor suppressor involved in cell proliferation and differentiation. The aim of this study was to determine the uterine expression pattern of this gene during early pregnancy in mice. Methods Uterine NDRG2 mRNA and protein expression levels were determined by RT-PCR and Western blot analyses, respectively, during the peri-implantation period in mice. Immunohistochemical (IHC) analysis was performed to examine the spatial localization of NDRG2 expression in mouse uterine tissues. The in vitro decidualization model of mouse endometrial stromal cells (ESCs) was used to evaluate decidualization of ESCs following NDRG2 knock down by small interfering RNA (siRNA). Statistical significance was analyzed by one-way ANOVA using SPSS 19.0 software. Results Uterine NDRG2 gene expression was significantly up-regulated and was predominantly localized to the secondary decidual zone on days 5 and 8 of pregnancy in mice. Its increased expression was associated with artificial decidualization as well as the activation of delayed implantation. Furthermore, uterine NDRG2 expression was induced by estrogen and progesterone treatments. The in vitro decidualization of mouse ESCs was accompanied by up-regulation of NDRG2 expression, and knock down of its expression in these cells by siRNA inhibited the decidualization process. Conclusions These results suggest that NDRG2 might play an important role in the process of decidualization during early pregnancy.


Background
Successful embryo implantation is a critical step in the establishment of pregnancy. Failure of implantation can lead to placental dysfunction, resulting in fetal retardation, preeclampsia (PE), and recurrent miscarriages [1,2]. A series of complex physiological events occur during the exquisitely regulated process of implantation, including blastocyst migration, apposition and adhesion to the luminal epithelium, extensive degradation and remodeling of the extracellular matrix, and invasion of trophoblast cells into the maternal endometrium, as well as decidualization of endometrial stromal cells (ESCs) [3,4]. Decidualization, characterized by proliferation and differentiation of ESCs, is a postovulatory process of endometrial remodeling that occurs in preparation for pregnancy. In addition, during pregnancy, the maternal decidua is essential for the establishment and maintenance of immuno-tolerance at the feto-maternal interface, and it regulates placental function and development of the conceptus [5,6]. However, the exact molecular mechanism underlying decidualization is still unclear. The identification of the factors involved in decidualization would undoubtedly contribute to a better understanding of the implantation process.
N-myc, which belongs to the Myc family, plays an essential role in cell proliferation and differentiation. Although N-myc has been well recognized for its oncogenic biofunction, it has been recently reported to exhibit a dynamic expression pattern during mouse embryonic development [7]. N-Myc downstream-regulated gene 2 (NDRG2), a member of the N-Myc downstream-regulated gene family, encodes a 357-amino acid protein with an apparent molecular weight of 41 kDa [8]. Consistent with its role in cellular differentiation and the stress response, NDRG2 has been identified as a tumor suppressor. Specifically, its expression has been shown to be decreased in a number of different cancer tissues, such as breast [9], liver [10] and gastric cancer tissues [11]. Its expression has also been detected in mouse embryos of various gestational ages. Further, during mouse fetal development, NDRG2 has been found to be strongly expressed in various tissues and organs [12], suggesting its potential role in embryonic development. This gene is phosphorylated by protein kinase B (PKB/Akt), PKC [13], and glucocorticoid-induced kinase 1 (SGK1) [14]. Akt/PKC signaling is a key regulator of trophoblast function during implantation as well as during early placentation [15]. Thus, we hypothesized that NDRG2 might be involved in the process of embryo implantation, and the present study was undertaken to examine the uterine expression pattern of this gene during the estrous cycle and peri-implantation period in mice.

Animals and tissue preparation
Adult ICR mice aged 8-10 weeks were obtained from the SIPPR/BK Laboratory Animal Company (Shanghai, China) and were caged at a controlled temperature (22°C) under a 14 h light: 10 h dark photoperiod. All experiments were conducted in full compliance with standard laboratory animal care protocols that were approved by the Institutional Animal Care Committee of Shanghai Institute of Planned Parenthood Research (Approval: 2013-19). The estrous cycle was staged by examining vaginal smears as previously described [16], and subsequently the uterine horns were removed from adult females at the diestrus, proestrus, estrous, and metestrus stages (n = 3, per stage) immediately after they were sacrificed by cervical dislocation.
Adult female mice were mated with fertile males of the same strain to achieve pregnancy (day 1 = day of vaginal plug). Pregnancy was confirmed on days 1 and 4 by recovering embryos from the reproductive tracts. Trypan blue dye solution (0.1 % in saline (w/v), 0.1 ml per mouse, Sigma) was injected via the tail vein on day 5 to visualize the implantation sites. The entire uterine horn was collected from the pregnant mice on days 1 and 4 of pregnancy (n = 3, per day). Uterine tissues at the implantation sites (IS) and non-implantation sites (NI) were separately collected from the pregnant mice on days 5 to 8 of pregnancy (n = 3, per day).
Pseudopregnant mice were obtained by mating adult females with vasectomized adult males. Artificial decidualization was induced by intraluminally infusing 25 μl of sesame oil into one uterine horn on day 4 of pseudopregnancy (n = 3), and the contralateral un-injected horn served as a control (n = 3). The mice were sacrificed at 72 h after decidualization was artificially induced [20]. Decidualization was confirmed by both weighing the uterine horns and histological examination of the uterine sections.
In the delayed embryo implantation model, pregnant female mice were bilaterally ovariectomized under ether anesthesia at 08:30-09:00 h on day 4 of pregnancy. The animals in the delayed embryo implantation group and activation group were subcutaneously injected with P 4 (1 mg/ 25 g body weight) dissolved in corn oil at 10:00 h from days 4 through 7 of pregnancy to maintain delayed implantation. Then, the animals in the activated implantation group (n = 3) received E 2 (25 ng/25 g body weight) along with P 4 to activate embryo implantation [20,21]. The female mice were euthanized at 10:00 h on day 8 of pregnancy, and the embryos were verified as previously described [22]. Delayed or activated implantation was confirmed by microscopic observation of the metabolically dormant or activated blastocyst in the uterine flush, as previously described [20]. Progesterone keeps the blastocyst dormant so that the inner cellular mass (ICM) remains underdeveloped. However, when estradiol is supplied with progesterone, the blastocyst becomes active, and the ICM becomes more prominent and distinct in appearance [20,23,24]. The uterine tissues were collected and then processed for RNA extraction and immunohistochemistry.

Primary culture of endometrial stromal cells and induction of decidualization in vitro
The isolation and culture of mouse ESCs was performed following previously described methods with minor modifications [25]. In summary, uterine horns were collected from pregnant mice on day 4 and cleaned to remove fat tissues. They were then slit longitudinally and washed thoroughly in Hanks' balanced salt solution (HBSS, Invitrogen, Carlsbad, CA) containing 100 U/ml penicillin (Invitrogen) and 100 μg/ml streptomycin (Invitrogen). Next, the tissues were placed in HBSS containing 10 mg/ml trypsin (Sigma), 6 mg/ml dispase (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin for 1 h on ice, followed by incubation for 1 h at room temperature and 10 min at 37°C. Following the digestion steps, the tissues were gently mixed, and the supernatant was discarded to remove the endometrial epithelial clumps. The partially digested tissues were then washed twice in HBSS and placed into HBSS containing 0.15 mg/ml collagenase (Invitrogen) at 37°C for 30 min. Following digestion and shaking, the contents of the tube were passed through a 70 μm gauze filter (Millipore, Darmstadt, Germany) to eliminate epithelial sheets. The cell pellets were washed twice and added to Dulbecco's modified Eagle's Medium-F12 medium (DMEM/F12) containing 10 % charcoalstripped fetal calf serum (FBS, Invitrogen) and antibiotics at 2 × 10 5 cells per well in a 6-well cell culture plate (Invitrogen). After incubation for 1 h, unattached cells were removed by several washes with HBSS, and cell culturing was continued after the addition of fresh DMEM/ F12 containing 1 % charcoal-stripped FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 nM E 2 , and 1 μM P 4 to induce decidualization of the ESCs.

siRNA transfection
NDRG2-targeting siRNAs were purchased from Santa Cruz Biotechnology, Inc. (NDRG2 siRNA (m) sc-40758, and sc-3700 7was used as the irrelevant control siRNA, Santa Cruz, Santa Cruz, CA). Prior to the in vitro decidualization of ESCs, NDRG2 siRNAs and control siRNAs were transfected into cultured ESCs according to the siPORT™ NeoFX™ protocol (Ambion/Life Technologies, Grand Island, NY). Briefly, 4 μl of siPORT NeoFX transfection reagent was mixed with 100 nM of siRNA duplexes to form complexes, and this mixture was then dispersed into each well of a 6-well cell culture plate.

Immunohistochemistry and histological analyses
Uterine tissues were fixed in freshly prepared 4 % buffered paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for over 40 h. Then, the tissues were dehydrated in graded alcohol and embedded in paraffin (Leica, Wetzlar, Hessen, Germany). Sections of uteri were processed for immunohistochemical detection. Briefly, the sections (5 μm) were deparaffinized and rehydrated in xylene and a graded series of ethyl alcohol, respectively, and then rinsed in PBS. Antigen retrieval was performed by placing the slides in boiling citric acid buffer (10 mmol/l of citrate sodium and 10 mmol/l of citric acid) for 15 min. The sections were cooled to room temperature and sequentially incubated at room temperature with 3 % hydrogen peroxide (H 2 O 2 ) in methanol for 15 min to quench endogenous peroxidases. The sections were then incubated with a rabbit anti-NDRG2 primary antibody (Abcam, Cambridge, UK) overnight at 4°C. After being washed with PBS, the sections were incubated with a biotinconjugated donkey anti-rabbit secondary antibody (1:200 in blocking solution, Proteintech Company, Wuhan, China). After another wash in PBS, they were incubated with peroxidase-conjugated streptavidin (1:200 in blocking solution, Proteintech Company) for 2 h. Then, they were stained with DAB (DAB, Zhongshan Corp., Beijing, China) according to the manufacturer's protocol and counterstained with hematoxylin (Sigma). For the negative controls, 10 % donkey serum was used instead of primary antibody. All the sections were examined and photographed under a microscope (DFC420C, Leica).
For vimentin and cytokeratin detection, cultured ESCs were washed in PBS and fixed with PBS containing 4 % paraformaldehyde for 20 min. After being washed in PBS, the cells were incubated in blocking buffer (0.2 % Triton-X100 and 10 % BSA in PBS) for 1 h. Next, they were incubated with blocking buffer containing an antivimentin mouse monoclonal antibody (1:100 dilution, Cell Signaling, Beverly, MA), anti-cytokeratin 8 mouse monoclonal antibody (1:50 dilution, Cell Signaling), or negative control mouse IgG antibody at 4°C overnight. After another wash in PBS, the cells were incubated in blocking buffer containing a biotin-conjugated donkey antirabbit antibody (1:200 in blocking solution, Proteintech Company) for 30 min. Then, the cells were again washed in PBS and incubated with peroxidase-conjugated streptavidin (1:200 in blocking solution, Proteintech Company) for 2 h. Next, the samples were stained with DAB (DAB, Zhongshan Corp.) according to the manufacturer's protocol. Finally, after being washed and stained with hematoxylin, the cells were observed and photographed under a microscope (DFC420C, Leica).

Western blotting
The collected uterine tissues were quickly frozen in liquid nitrogen and granulated into a fine powder. The tissue powder was homogenized in lysis buffer (Beyotime, China). Then, the tissue lysate was centrifuged, and the supernatant was transferred into a new tube. Cultured ESCs were collected in lysis buffer, and the lysate was centrifuged to collect the supernatant. Protein concentrations were measured by Bradford assay (Bio-Rad, Hercules, CA), and 50 μg of total protein was separated on a 12 % acrylamide gel and then transferred electrophoretically onto nitrocellulose membranes (Millipore). The membranes were incubated overnight at 4°C with specific primary antibodies against NDRG2 and beta-actin (Santa Cruz), followed by incubation with the appropriate secondary antibodies. The blot was developed using a PhosphaGLO AP Substrate Kit (KPL, Gaithersburg, MD) according to the manufacturer's protocol. All samples were analyzed by Western blot in triplicate. Band intensities were quantified by densitometry using ImageJ software (U.S. National Institutes of Health, MD).

Statistical analysis
All values were presented as the mean ± SEM, as determined from at least three independent experiments. Statistical significance was assessed by one-way ANOVA. A P < 0.05 was considered statistically significant. Statistical analysis was conducted using SPSS 19.0 software (SPSS Software, Chicago, IL).

Results
Uterine NDRG2 expression during the estrous cycle and peri-implantation period The uterine expression patterns of NDRG2 mRNA and NDRG2 protein during the mouse estrous cycle were determined by real-time PCR and IHC, respectively. The results showed distinctive NDRG2 protein signals in luminal and glandular epithelial cells at the diestrus (Fig. 1a), proestrus (Fig. 1b), estrous (Fig. 1c) and metestrus phases (Fig. 1d), with the most prominent expression detected during the estrous phase, along with weak positive signals in stromal cells (Fig.1c). Consistently, real-time PCR analysis revealed that NDRG2 mRNA expression was significantly increased during the estrous phase compared with that during the diestrus, proestrus and metestrus phases (Fig. 1e).
Prior to the initiation of implantation, uterine NDRG2 mRNA expression gradually increased from day 1 (D1) to day 4 (D4) of pregnancy. During implantation (day 5 to day 8 of pregnancy), NDRG2 mRNA expression was differentially regulated between the implantation sites and non-implantation sites. Its expression was significantly up-regulated at the implantation sites, whereas it was obviously down-regulated at the non-implantation sites (Fig. 2a). Meanwhile, the results of Western blot analysis showed that uterine NDRG2 protein expression was significantly increased after the initiation of implantation (day 5 of pregnancy), and its expression at the implantation sites was remarkably higher than that at the non-implantation sites (Fig. 2b). Furthermore, the spatiotemporal expression of NDRG2 protein in the mouse uterus during early pregnancy was examined by IHC. There was no detectable NDRG2 protein signal in the uterine tissue obtained from pregnant mice on day 1 (Fig. 2c/b) or day 3 (Fig. 2c/c). On day 4 of pregnancy, faint NDRG2 protein signals were noted in the subepithelial stromal bed (Fig. 2c/d). On day 5 of pregnancy, the distinct accumulation of NDRG2 protein signals was observed in both the secondary decidual zone and the embryo, with faint expression in the primary decidua immediately adjacent to the implanting embryo ( Fig. 2c/e, f). The distribution of NDRG2 protein signals on day 8 of pregnancy was similar to that on day 5, except that the positive signals also accumulated in trophoblasts at implantation sites ( Fig. 2c/g, h).

The effects of steroid hormones on NDRG2 expression in the uterus
Ovariectomized mice were used to examine the effects of steroid hormones on NDRG2 expression in the uterus. Compared with the control (Fig. 3a), treatment with E 2 (Fig. 3b) or P 4 (Fig. 3c) significantly induced NDRG2 protein expression in both the luminal and glandular epithelia, and the combined administration of E 2 and P 4 resulted in a similar effect on its expression (Fig. 3d). A similar alteration in NDRG2 mRNA expression was observed following treatment with E 2 or P 4 (Fig. 3e).

The association of uterine NDRG2 expression and artificial decidualization and activated implantation
The artificial decidualization model was used to examine whether uterine NDRG2 expression is dependent on the presence of a living embryo or whether it is induced by the decidualization reaction alone. The results of IHC analysis showed strong NDRG2 protein signals in decidualized stromal cells (Fig. 4b), whereas no visible signals were found in control uteri (Fig. 4a). Meanwhile, uterine NDRG2 mRNA expression was also found to be significantly up-regulated by artificial decidualization (Fig. 4c).
In the delayed implantation model, the NDRG2 protein was abundantly expressed in activated implantation uteri (Fig. 4e), and it was weakly expressed in delayed implantation uteri (Fig. 4d). Likewise, significantly higher NDRG2 mRNA levels were detected in the activated implantation uteri compared with those in the delayed uteri (Fig. 4f).

NDRG2 expression in cultured ESCs during the process of in vitro decidualization
To explore the function of NDRG2 in the decidualization of mouse ESCs, a mouse primary ESCs culture system was established. Positive staining for vimentin and negative staining for cytokeratin were considered to be indicative of ESC purity (Fig. 5a). The transformation of decidual cells from ESCs was indicated by DTPRP mRNA expression [26] (Fig. 5b). The mRNA and protein levels of NDRG2 were found to be significantly increased during in vitro decidualization (Fig. 5c, d).

The effects of decreased NDRG2 expression in ESCs on in vitro decidualization
To further explore the role of NDRG2 in the decidualization of ESCs, NDRG2 expression was knocked down in cultured ESCs using targeting siRNAs. As a result, its expression was reduced by more than 60 % in NDRG2 siRNA-transfected ESCs compared with that in control ESCs (Fig. 6a, c, d), and this reduction was correlated with a significant decrease in DTPRP mRNA expression (Fig. 6b), indicating that down-regulation of NDRG2 expression inhibited the decidualization of mouse ESCs.

Discussion
The present study demonstrated the expression pattern of NDRG2 in the uterus during the estrous cycle and early pregnancy in mice. Uterine NDRG2 expression was found to peak during the estrous phase and to be induced by the ovarian steroid hormones E 2 and P 4 . During early pregnancy, uterine NDRG2 expression was significantly increased at implantation sites, with predominant localization in the decidual zone. This increase in expression was accompanied by the activation of delayed implantation, as well as artificially induced decidualization, both in vivo and in vitro. Furthermore, down-regulation of NDRG2 expression in mouse ESCs significantly inhibited decidualization in vitro.
The synergistic actions of E 2 and P 4 are critical for regulation of the estrous cycle and establishment of uterine receptivity [27,28]. A putative estrogen-response element (ERE) is located in the promoter region of the NDRG2 gene, and NDRG2 expression in astrocytes is regulated by estrogen [29,30]. In the present study, we found that both E 2 and P 4 had stimulatory effects on uterine NDRG2 expression in ovariectomized mice (Fig. 3). In addition, during the normal estrous cycle, uterine NDRG2 expression was significantly increased during the proestrus and estrous phases, whereas it was decreased during the metestrus and diestrus phases (Fig. 1), consistent with the E 2 surge that takes place during the proestrus and estrous phases [31]. Because NDRG2 expression was detected in luminal and glandular epithelial cells as well as in stromal cells, we speculated that it might participate in regulating cyclic proliferation and differentiation in endometrial epithelial/stromal cells to optimally prepare for embryo implantation under the control of E 2 and P 4 during the estrous cycle.
Successful embryo implantation is integral to the establishment of pregnancy, and initiation of implantation coincides with the establishment of uterine receptivity and the subsequent decidualization of endometrial stromal cells (ESCs) [32]. In the mouse, a pre-ovulatory E 2 surge stimulates uterine epithelial cell proliferation on day 1 of pregnancy, followed by a rise in the P 4 level and its secretion by the newly formed corpora lutea, which initiates uterine stromal cell proliferation on day 3. Embryos enter the uterus at midnight on day 3 or in the early hours of day 4 after the uterus is exposed to an Fig. 2 NDRG2 expression in the mouse uterus during early pregnancy. a Quantitative PCR analysis of NDRG2 mRNA expression in the uterus during early pregnancy (n = 3, *P < 0.05). b Western blot and densitometric analyses of uterine NDRG2 protein levels during early pregnancy (n = 3). All experiments were repeated three times. The data are shown as the mean ± SEM. *P < 0.05. c Immunohistochemical analysis of uterine NDRG2 protein expression on days 1, 3, 4, 5, and 8 of pregnancy. IS, implantation sites; NI, non-implantation sites. *, indicates the location of the embryo. le, luminal epithelium; ge, glandular epithelium; st, stroma; pdz, primary decidual zone; sdz, secondary decidual zone; D, day of pregnancy; gc, giant cell; Scale bar represents 100 μm increased concentration of P 4 for at least 24 h on day 3 followed by exposure to estrogen, causing it to be receptive for embryo implantation [33]. We found that uterine NDRG2 expression remained increased during the preimplantation period from day 1 to day 4, suggesting its potential role in establishing uterine receptivity under the regulation of ovarian steroids.
Once mouse embryos attach to a receptive endometrium, decidualization is triggered by extensive proliferation and differentiation of ESCs into decidual stromal cells (DSCs) at embryo implantation sites. Because the decidual response can be induced in a reproducible manner in the absence of an embryo and decidual zones are easily discernable, the mouse is a good model to investigate the mechanism of decidualization. In the mouse, at least three factors may be necessary for normal decidualization, including E 2 , P 4 and embryonic or physical stimulation (by intra-luminal infusion of oil or scratching with a needle). Increases in E 2 and P 4 stimulate the proliferation and differentiation of ESCs surrounding invading trophoblast cells to support the decidualization process [33][34][35]. In the present study, a significant increase in NDRG2 expression was observed at implantation sites on days 5 and 8 of pregnancy in the mice, and NDRG2 protein signals were predominantly localized to the decidual zone. Moreover, because E 2 and P 4 induced NDRG2 expression in the uterus and increased its expression in ESCs following artificial decidualization  Fig. 5), we hypothesized that it might be involved in the decidualization of these cells. To further investigate this hypothesis, we specifically knocked down NDRG2 expression in mouse ESCs using NDRG2targeting siRNAs and subsequently evaluated the effect of decreased NDRG2 expression on ESC decidualization in vitro using DTPRP as a marker of decidualization. Decreased expression of this gene was found to remarkably inhibit decidualization in vitro (Fig. 6), indicating that it may participate in this process.
An implantation site is distinguished from a nonimplantation site not only by the presence of DSCs and trophoblast cells but also by the enrichment of decidual immune cells. Dendritic cells (DCs) are the most predominant immune cells in decidua at implantation sites, and they play a critical role in preserving a hospitable microenvironment for pregnancy [36]. Although NDRG2 expression in decidual DCs has not been reported, its expression in DCs derived from CD 34+ progenitor cells has been detected. Inhibition of DCs differentiation is accompanied by reduced NDRG2 expression, and the down-regulation of its expression negatively affects the ability of DCs to stimulate T cells proliferation [37]. Thus, an increased in NDRG2 expression at implantation sites during early pregnancy might also be associated with the differentiation and maturation of decidual DCs. NDRG2 expression in decidual DCs will be explored further by our group.
Although the exact role of NDRG2 in regulating ESCs and DSCs needs to be further explored, NDRG2 has been reported to contain several potential phosphorylation sites and to be phosphorylated by Akt [13] and SGK1 [14]. Importantly, a decrease in uterine Akt expression has been shown to result in abnormal decidualization in mice [38], and reduced SGK1 expression in DSCs has been observed in recurrent spontaneous miscarriage (RSM) patients [39]. Thus, it is reasonable to believe that the function of NDRG2 in ESCs and DSCs might be partially regulated by the Akt or/and SGK1 signaling pathway(s). Conversely, the appropriate invasion of trophoblast cells into maternal stroma is also a pivotal event that occurs during implantation [40]. Because we observed NDRG2 protein expression in trophoblast cells at implantation sites on day 8 of pregnancy in the mice in this study (Fig. 2) and it has been demonstrated that E 2 and P 4 participate in the regulation of trophoblast cells [41,42], we hypothesize that NDRG2 might also be involved in the invasion of trophoblast cells during early pregnancy.

Conclusions
In summary, the present study has demonstrated that steroid hormones stimulate NDRG2 expression in the uterus and that NDRG2 expression is significantly upregulated at implantation sites during early pregnancy in mice. During in vivo and in vitro artificial decidualization, NDRG2 expression was found to be remarkably increased. Further, down-regulation of its expression in ESCs inhibited in vitro decidualization. These results suggest that NDRG2 might be essential for embryo implantation and decidualization.