Dicer-deficient mice (129/Ola and C57BL/6 mixed background), obtained using a gene-trap method, were described previously (13
). The mice used in this study were female offspring littermates derived from crosses between male and female Dicer+/d
mice after more than 10 generations of sibling breeding (13
). All experiments were carried out in compliance with the rules of, and were approved by, the Animal Use Committee of The Scripps Research Institute.
Timed mating experiments.
For timed mating experiments, Dicer+/+ and Dicerd/d female mice were bred with fertile wild-type males. To induce superovulation, mice were first injected (i.p.) with pregnant mare’s serum gonadotropin (5 IU/mouse; Sigma-Aldrich) and then with human chorionic gonadotropin (5 IU/mouse; Sigma-Aldrich) 48 hours later. The female mice were then bred with male mice overnight. The following morning (day 0.5 after coitus), the female mice were checked for a vaginal plug. The mice were sacrificed on day 1.5. Oocytes and 2- and 4-cell stage embryos, collected by flushing the oviducts with PBS, were examined under a dissecting stereomicroscope.
Ovary transplantation was performed as described previously (45
) using 4- to 6-week-old female littermate mice to avoid immunological rejection of tissues. After anesthesia with ketamine, a dorsolateral incision about 1.0 cm long was made in the lumber region on each side of the midline. Through the incision, the ovaries were gently pulled outside the body. The ovarian bursa was cut to make a small incision at the opposite side of the oviduct. The ovaries were removed by cutting the ovarian stalk with scissors. After the bleeding stopped, donor ovaries were transplanted into the empty bursas of each recipient mouse. The bursas were placed back, and the reproductive tract was returned to the abdominal cavity. The skin incision was closed with sutures. Two weeks after the operation, treated mice were mated with fertile male mice. The genotype of the offspring born to the recipient mice was confirmed by PCR (13
Histological analysis, immunohistochemistry, and immunofluorescence.
Tissues were dissected out, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and analyzed by H&E staining. For type IV collagen immunofluorescent studies, sections were stained with primary anti–type IV collagen (Abcam) and secondary Alexa Fluor 488–conjugated goat anti-rabbit IgG antibodies (Molecular Probes). For CD31/PECAM immunofluorescent studies, frozen sections were incubated with primary anti-CD31/PECAM (MEC 13.3; BD Pharmingen) and then with secondary Alexa Fluor 594–conjugated goat anti-rat IgG antibodies (Molecular Probes). Slides were mounted using VectaShield (Vector Labs). Cumulative vessel length in CLs was evaluated as the average of the number of vessels per 100 × 100 μm2 multiplied by the vessel length in 3 random fields at ×400 fields from 4 different pairs.
Angiogenesis-related protein antibody array.
Mouse Lysate Angiogenesis Antibody Array, which detects 24 different kinds of angiogenesis-related cytokines, was purchased from Chemicon. Dicer+/+ and Dicerd/d mouse ovaries, day 1.5 after coitus, were used for the screening according to the manufacturer’s instructions. The data were evaluated using FluorChem8900 software (Alpha Innotech). A grid containing a series of circles corresponding to the spots of the array was laid over the image, and the circles were adjusted to each spot. The intensities within the circle were calculated, and the local background intensities around the circle (intensities in the outer torus with 1.3-fold longer diameter of the spot circle) were subtracted to reduce bias. Genes whose signal intensities were lower than 5 times their background intensities were excluded from further analyses to exclude the variability due to their low expression levels. The normalized signal intensities of each gene were determined as the intensities divided by the average intensities of positive control spots on the same array. Relative protein expression levels between different samples were determined as signal intensity ratios, obtained by dividing the normalized signal intensities of each gene from different samples. Because each gene has 2 spots on one array, 4 values for each gene derived from 2 independent experimental sets were used for one statistical analysis. A scatter plot was depicted using log10 conversion of the average of normalized signal intensities of each gene.
Primer extension was performed as described previously (5
). The primer sequences of miR17-5p and let7b are ACTACCTGCACTGTAAGC and AACCACACAACCTACTAC, respectively.
Prediction of miRNA target sites.
Two computational miRNA target prediction programs, MicroInspector (34
) and miRanda (36
), were used to predict miRNA binding sites in 3′-UTR mTIMP-1 sequences. MicroInspector was run with the hybridization temperature set to 37°C, and the free-energy cutoff was set to –22 kcal/mol. Of the primary candidate miRNA sequences, those miRNAs that were confirmed by another program as rna22
) were selected as candidates. miRanda was run according to the authors’ recommendation.
Reporter plasmids and reporter assay.
The vector pSPORT1-mTIMP1, which contains full open reading frame sequences of mTIMP1 (IMAGE ID: 30056565), was purchased from ATCC. The reporter plasmid was constructed by subcloning PCR-amplified 3′-UTR fragments from the pSPORT-mTIMP1 into the downstream of a Photinus pyralis (firefly) luciferase reporter gene using EcoR1 and Kpn1 sites. To check the transfection efficiency, pRL-TK, a control plasmid (Promega), was used. Transfection was performed using Lipofectamine 2000 (Invitrogen). Luciferase assays were carried out with the Dual-Luciferase Reporter Assay System (Promega).
pcDNA3-TIMP1 expression vector was constructed by subcloning PCR-amplified coding sequence fragments from the pSPORT-mTIMP1 with EcoR1 and Kpn1 sites. pSuper-miR378, pSuper–miR17-5p, and pSuper-let7b, which express miR378, miR17-5p, and let7b, respectively, were constructed by cloning the miR sequences into the pSuper vector using XhoI and BglII sites.
Western blotting analysis and antibodies.
The cell extracts and homogenized mouse tissues were normalized for protein concentration using the Bio-Rad Dc Protein Assay Kit (Bio-Rad), or, for the detection of TIMP1 in the overexpression of miRNA, cell extracts were normalized based on the luciferase value of cotransfected pRL-TK to avoid the variances of transfection efficiency. Protein (30 μg) was resolved by SDS-PAGE, transferred to PVDF (Hybond-P; Amersham Pharmacia Biotech), and immunoblotted. A goat polyclonal antibody against mTIMP1 was purchased from R&D systems. A rabbit monoclonal anti-VEGF receptor 2 antibody (55B11) was purchased from Cell Signaling. A mouse monoclonal anti-VEGF receptor 1 (Flt-1) antibody (MAB1664) and a mouse anti-GAPDH antibody were purchased from Chemicon. The bound antigens were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).
The mouse endothelial cell line SVEC4-10 and the human embryonic kidney cell line 293T were obtained from ATCC and cultured in DMEM supplemented with 10% FBS.
SVEC or 293T cell culture supernatant after transfection of the TIMP1 expression vector with miRNA expression plasmids anti-miRNA oligos, or miRNA precursors were analyzed for the activity of TIMP1 and MMP2 as described previously (46
). Because MMPs are separated from their inhibitor TIMP1 during the normal zymographic procedure, their activities cannot be analyzed accurately. To avoid this problem and better define the balance existing between MMP2 and its inhibitor TIMP1, we measured the TIMP1 activity by reverse zymography. Briefly, 36 hours after transfection (control antisense oligos with random sequences, anti–miR17-5p and let7b oligonucleotides, control miRNA precursors with random sequences, or double-stranded miR17-5p and let7b precursors), supernatants were collected, concentrated 10-fold using the Centricon concentrator (Amicon), and electrophoresed on nondenaturing 0.1% SDS, 12% polyacrylamide gels containing 1 mg/ml gelatin, and 0.16 μg/ml 4-aminophenylmercuric acetate–activated MMP-2 (both from EMD). Electrophoresis was carried out at 4°C, and then the gel was renatured in 2.5% Triton X-100 for 3 hours with 3 times replacement of fresh solution at room temperature. Then, the gel was incubated in enzyme buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35) for 24 hours at 37°C and stained with Coomassie blue G-250. The gel image and the integrated intensities were obtained and quantified using FluorChem8900.
Endothelial tube formation assay, cell proliferation assay, and migration assay.
Anti–miRNA17-5p and let7b oligonucleotides, double-stranded miRNA17-5p, and let7b precursors and random-sequence anti-miRNA oligos and miRNA precursors were purchased from Ambion; 40 nM anti-miRNA oligos or 40 nM miRNA precursors were transfected into SVEC. Control antisense oligos with random sequences and control miRNA precursors with random sequences were used as negative controls. Thirty-six hours after transfection, 1 × 105 cells were seeded in a 12-well plate coated with 200 μl Growth Factor Reduced Matrigel (BD Biosciences). Tube length was quantified after 12 and 18 hours by measuring the cumulative tube length in 3 random microscopic fields with a computer-assisted microscope using the program AxioVision 3.1 (Carl Zeiss). For the cell proliferation assay, 36 hours after transfection, 5 × 104 cells were seeded and the number of cells was determined by cell counting at the indicated time points. For the examination of cell motility, an in vitro wound healing assay was performed. Transfected cells were seeded with 2.5 × 106 cells in a 12-well plate and cultured in 1% FBS for 24 hours to synchronize and were then wounded by removing a 200- to 400-mm strip of cells across the well with a 200-μl pipette tip. Wound healing was quantified as the average length of the elongation of wound edges over 24 hours by using AxioVision 3.1.
Serum progesterone levels were measured in serum samples collected at indicated days after coitus using a rodent progesterone ELISA kit according to the manufacturer’s instruction (Endocrine Technologies Inc.). The limit of sensitivity of the assay was 0.1 ng/ml.
Semiquantitative RT-PCR analysis.
Total RNA was extracted from ovaries using Trizol reagent (Invitrogen), and RT-PCR was done as described previously (13
). The primers used were as follows: cytochrome P450 family 11 subfamily a polypeptide 1, 5′-AGAAGCTGGGCAACATGGAGTCAG-3′ and 5′-TCACATCCCAGGCAGCTGCATGGT-3′; luteinizing hormone receptor, 5′-CTTATACATAACCACCATACCAG-3′ and 5′-ATCCCAGCCACTGAGTTCATTC-3′; prolactin receptor, 5′-ATACTGGAGTAGATGGGGCCAGGAGAAATC-3′ and 5′-CTTCCATGACCAGAGTCACTGTCAGGATCT-3′. The result was quantified using FluorChem software and normalized by GAPDH intensities.
miRNA injection technique.
Mice were anesthetized using ketamine. A dorsolateral small incision was made in the lumber region on each side of the midline. Through the peritoneal incision, the ovaries were gently pulled outside the body. The intrabursal injection was performed under microscopic magnification by inserting a 30-gauge needle through the ovarian fat pad into the ovarian bursa. The ovaries were returned to the abdominal cavity, and the wound was sutured with 4-0 silk (Ethicon Inc.). For histological analyses, 5 female Dicerd/d mice were used. In each mouse, a 50-μl mixture of 0.5 nmol double-stranded miRNA17-5p and let7b precursors with normal saline and lipofectamine 2000 was delivered into the right ovary, and transfection reagents alone were injected into the left ovary as a control. To examine the course of pregnancy, 5 Dicerd/d female mice were injected with miRNAs into both ovaries. To determine the injection and transfection efficiency, Cy-3–labeled, fluorescent siRNA (siGLO; Dharmacon) was injected in the same way. Injection of transfection reagent alone was used as control. Three days after the procedure, the ovaries were sectioned, frozen, and examined under a fluorescent microscope. Mice were initially housed separately; when they resumed normal behavioral activity, they were housed with a fertile male for mating. Vaginal plugs were confirmed the next morning (day 0.5 of pregnancy). For the histological analyses, mice were sacrificed on day 1.5, and the ovaries were sectioned, frozen, and then examined histologically. Western blotting of TIMP1 and the antibody analyses were performed using the lysates of control, and the injected ovaries were extracted on day 1.5 of pregnancy. For the analyses of serum progesterone levels, serum samples were collected on the indicated days of pregnancy from 4 Dicerd/d mice in which both ovaries had been injected. In the case of Dicer+/+ mice, the same methods were used, except that anti–miRNA17-5p and let7b oligos were used instead of miRNA precursors.
The statistical significance of any differences was determined using the 2-tailed Student’s t test or Welch’s t test when variances were unequal.