This study was approved by the Institutional Review Board of Pusan National University Hospital, Korea. All experiments with mice were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, approved by the Pusan National University Hospital Institutional Animal Care and Use Committee.
C57BL/6 inbred female mice were purchased from Korea Experimental Animal Center (Daegu, Korea). The mice were maintained on light(L)-dark(D) cycle with 14/10 h L/D with food and water available ad libitum.
Administration of BMP-6 during superovulation
Aged female mice of 26–31 weeks old were superovulated by intraperitoneal injection with 0.1 mL of 5 IU equine chorionic gonadotropin (eCG) (Sigma, St. Louis, MO, USA) containing various doses (0, 0.01, 0.1, 1, and 10 ng) of recombinant mouse BMP-6 (R&D Systems, Minneapolis, MN, USA), followed by injection of 5 IU of human chorionic gonadotropin (hCG, Sigma) approximately 48 hours later. Then the mice were immediately paired with an individual male that previously tested for fertility. The following morning the mice were inspected, and those with a confirmed vaginal plug were considered mated and fertilized. The aged control group was superovulated without BMP-6. Young mice of 6–9 weeks old were also superovulated without BMP-6 as a positive control for ovarian stimulation and in vitro culture of embryos.
Zygotes collection and in vitroculture of embryos
Eighteen hours after hCG injection, female mice with a confirmed vaginal plug were sacrificed by cervical dislocation. Cumulus-enclosed one-cell embryos (zygotes) were retrieved from the oviductal ampulae and denuded by incubation for 1 minute with 0.1% hyaluronidase (Sigma) in Dulbecco's phosphate buffered saline (dPBS; Gibco BRL, Grand Island, NY, USA). Zygotes retrieved from each were individually pooled and washed three times in P1 medium (Irvine Scientific, Santa Ana, CA, USA) with 10% serum substitute supplement (SSS; Irvine Scientific). All the zygotes except for those with cell fragmentation were cultured in 30 μl drops of P1 medium with 10% SSS for the first 2 days, and then blastocyst medium (Irvine Scientific) with 10% SSS for the later 2 days under paraffin-oil at 37°C in a 5% CO2 humidity incubator. The media were changed daily as 30 μL-drop culture. When the retrieved one cell embryos developed to 2-cells embryo in the first day of culture, we included the data in this study. However, unless all retrieved one cell embryos developed to 2-cell embryo, we excluded the data of the mouse without developed 2-cell embryos. In this respect, we defined the retrieved one-cell embryo as real zygotes.
Ovary collection and examination of ovarian Id-1 and VEGF expression
Just after the retrieval of the zygotes, both ovaries of each mouse were collected. For the examination of ovarian expression of Id-1 and VEGF, each ovary was randomly allocated to half for reverse transcriptase-polymerase chain reaction (RT-PCR), half for western blot and a whole for immunohistochemistry (IHC).
Total RNA was isolated from frozen tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendation. RNA was eluted in RNase-free water and stored at −80°C until RT-PCR analysis. The cDNA was synthesized from 5 μg of total RNA with AMV Reverse Transcriptase (Promega, Madison, WI, USA) using a random hexamer (Bioneer, Daejeon, Korea) at 42°C for 1 hour followed by inactivate the enzyme at 95°C for 5 minutes. Template cDNA was subjected to PCR amplification using gene-specific sense and antisense primers. PCR conditions were denaturation at 95°C for 30 seconds, specific annealing temperature for 30 seconds, and extension at 72°C for 30 seconds in a thermal cycle. The primers of each gene are as follows: 5’-CTGCTCTACGAC ATGAACGGCTG -3’ (sense) and 5’-CGACACAAGATGCGATCGTC -3’ (antisense) for Id-1 (32 cycles); 5’-CTTGTTCAGAGC GGAGAAAGC- 3’(sense) and 5’-ACATCTGCAAGTACGTTCGTT-3’ (antisense) for VEGF (40 cycles); and 5’-ACCACAGTCCATGCCATCAC-3’ (sense) and 5’-TCCACCACCCTGTT GCTGTA-3’ (antisense) for GAPDH (25 cycles). GAPDH mRNA was quantified in each sample as an internal control to normalize the level of mRNA among samples. The PCR products were examined by 2% agarose gel electrophoresis. Data are representative of at least three independent experiments. The relative density of PCR bands were quantified and normalized relative to the control band with the National Institutes of Health (NIH) Image program (Image-J 1.35d, NIH, Bethesda, MD, USA).
Western blot analysis
Protein was extracted by homogenization of ovaries in the presence of ice-cold lysis buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, and 1 mM EDTA) containing protease inhibitor. The protein content of the cell lysate was determined with Bradford reagent (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as the standard. Sixty micrograms of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was incubated with anti-Id-1 polyclonal antibody (1:200; Santa Cruz Biotech, Santa Cruz, CA, USA), anti-VEGF monoclonal antibody (1:100; R&D Systems) and anti-β-actin monoclonal antibody (1:5,000; Sigma) in tris-buffered saline (TBS) containing 1% tween 20 (TBS-T) supplemented with skim milk overnight at 4°C. After washing three times with TBS-T, the blotted membranes were incubated with horseradish peroxidase (HRP)-conjugated goat antibody (Santa Cruz Biotech) for 30 minutes at room temperature. After washing three times with TBS-T, the proteins bands were visualized using an enhanced chemiluminescence (ECL) detection system according to the recommended procedure (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Actin expression was used as the control. Data are representative of at least three independent experiments. The relative density of protein bands were quantified and normalized relative to the control band with the National Institutes of Health (NIH) Image program (Image-J 1.35d).
Immunohistochemistry was performed on 4 μm-thick, formalin-fixed paraffin sections of ovary tissues using Zymed's SuperPicTure™ Polymer detection system (Zymed Laboratories-Invitrogen, San Francisco, CA, USA). Serial sections of the ovary were mounted on coated slides and placed in an oven at 60°C for 1 hour. The slides were then deparaffinized in xylene and dehydrated in a graded series of ethanol. The slides were boiled in 10 mM citrate buffer (pH 6.0) for 15 minutes in a microwave oven. The endogenous peroxidase was quenched with 0.3% hydrogen peroxide at room temperature for 10 minutes, and then tissues were rinsed four times for 5 minutes each in PBS. The sections were incubated overnight at 4°C with the primary Id-1 antibody (Santa Cruz Biotech) and VEGF antibody (Lab Vision, Fremont, CA, USA) at 1:100 dilutions. The remaining steps were performed according to the instructions supplied with the kit. After washing three times with PBS, the samples were incubated with biotinylated-conjugated secondary antibody and HRP coupled to streptavidin-conjugated antibody for 15 minutes at room temperature and washed three times with PBS. The sections were stained with 3,3-diaminobenzidine (DAB), counterstained with Mayer’s hematoxylin (Sigma), and mounted with histomount solution (Invitrogen). The results were assessed by two pathologists using a light microscope.
An SPSS program (version 12.0) was used for statistical analysis, and all data were presented as a mean±SD. The number of zygotes retrieved and blastocyst formation rate according to treated BMP-6 concentration were analyzed by one-way analysis of variance with post hoc multiple comparisons by Bonferroni-Dunn analysis. Statistical analysis for comparison of expression of Id-1 and VEGF was performed by one-way ANOVA. A P value of < .05 was considered statistically significant.