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

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.

To observe the effects of ovarian steroid hormones on uterine NDRG2 expression, adult females were ovariectomized and allowed to rest for 2 weeks. Then, the ovariectomized mice were randomly divided into four groups (n = 3, per group) and injected with (1) sesame oil (0.1 ml/mouse), (2) 17β-estradiol (E₂, 100 ng/25 g body weight, Sigma, St. Louis, MO), (3) progesterone (P₄, 1 mg/25 g body weight, Sigma), or (4) E₂ (100 ng/25 g body weight) plus P₄ (1 mg/25 g body weight) according to previously described methods [17‐19]. Steroids were dissolved in sesame oil and injected subcutaneously at the same volume (0.1 ml/mouse). The mice were sacrificed at 24 h after treatment, and the uterine horns were collected.

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 P₄ (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 E₂ (25 ng/25 g body weight) along with P₄ 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.

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 % charcoal-stripped fetal calf serum (FBS, Invitrogen) and antibiotics at 2 × 10⁵ 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 E₂, and 1 μM P₄ to induce decidualization of the ESCs.

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.

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 (H₂O₂) 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 biotin-conjugated 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 anti-vimentin 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 anti-rabbit 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).

Total RNA was extracted from uterine tissues or ESCs using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Extracted RNA was dissolved in diethylpyrocarbonate (DEPC, Sigma)-treated water, and the RNA concentration and purity were estimated by measuring absorbance at 260 and 280 nm with a NanoDrop 2000 (Thermo Scientific, Waltham, MA). cDNAs was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI), according to the manufacturer’s instructions. Real-time PCR was performed in a 20 μl reaction volume using an ABI 7500 thermal cycler (Applied Biosystems, Foster City, CA). The thermal cycling conditions were 95 °C for 30 s, followed by 40 cycles at 94 °C for 5 s and 60 °C for 30 s. Melt curve analysis and agarose gel electrophoresis were then conducted to monitor the purity of the PCR products. Beta-actin was used as an endogenous control. The following primers were used: NDRG2, sense, 5′-AGAACTTCGTGCGGGTTCAT-3′, antisense, 5′-TCGCGACAGAATGTAGGCTC-3′; decidual/trophoblast PRL-related protein (DTPRP), sense, 5′-AAGAATGCCCTTCAGCGAGC-3′, antisense, 5′-AGCTGGTGGGTTTGTGACAT-3′; and beta-actin, sense, 5′-GGCTGTATTCCCCTCCATCG-3′, antisense, 5′-CCAGTTGGTAACAATGCCATGT-3′.

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).