General chemicals, reagents cells and animals. Chemicals and reagents were obtained
from Sigma-Aldrich; Merck KGaA unless otherwise stated. The NIH3T3 cell line was purchased from the American Type Culture Collection. A total of 265 3-week-old female specific pathogen free ICR mice (weighing 18-20 g) used in this study were obtained from Vital River Experimental Animal Technical Co., Ltd. Animals were housed at a temperature of 20-26˚C and a humidity of 40-70% with a 12 h light/dark cycle. The mice were fed in feeding boxes, and the frequency of food replacement was 2 times a week, and the frequency of water bottle replacement was 3 times a week. All animal experiments were approved by the Animal Care and Use Committee of Nanjing Medical University (Nanjing, China) and performed in accordance with institutional guidelines.
Antibodies. Mouse monoclonal anti-β-actin (cat. no. A5316-100) antibody was obtained from Sigma-Aldrich; Merck KGaA. Mouse monoclonal anti-SPASTIN (A-4) (cat. no. sc-398264) and mouse monoclonal anti-β-tubulin ( cat. no. sc-5274) antibodies were purchased from Santa Cruz Biotechnology, Inc. Human anti-centromere CREST antibody (cat. no. 15-234) was purchased from Antibodies Incorporated. Rabbit polyclonal anti-CRMP5 (cat. no. ab36203) was purchased from Abcam. Cy2-conjugated donkey anti-mouse IgG (code no. 715-225-150), rhodamine (TRITC)-conjugated donkey anti-goat IgG (code no. 705-025-147), and Alexa Fluor 647-conjugated donkey anti-human IgG (code no. 709-605-149) were purchased from Jackson ImmunoResearch Laboratories, Inc. Horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (cat. no. 31402) and HRP-conjugated goat anti-mouse IgG (cat. no. 31430) were purchased from Invitrogen; Thermo Fisher Scientific, Inc.
Oocyte collection and culture. Immature oocytes arrested in prophase I [germinal vesicle (GV) oocytes] were obtained from the ovaries of 3-week-old female ICR mice. The mice were first euthanized with CO2 and then sacrificed by cervical dislocation, and the ovaries were isolated and placed in operation medium (HEPES) with 2.5 nM milrinone and 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.). Oocytes were released from the ovary by puncturing the follicles with a hypodermic needle. Cumulus cells were washed off the cumulus-oocyte complexes, and 50 isolated denuded oocytes were placed in 100-µl droplets of culture medium under mineral oil in plastic dishes (BD Biosciences). The culture medium was MEM+ (MEM with 0.01 mM EDTA, 0.23 mM Na-pyruvate, 0.2 mM pen/strep, 3 mg/ml BSA and 20% FBS). The oocytes were cultured at 37.0°C, 5% O2, and 5% CO2 in a humidified atmosphere. Prior to in vitro maturation (IVM), all culture medium included 2.5 nM milrinone to prevent the resumption of meiosis.
siRNA production and microinjection. Sequences of all DNA templates used for siRNA production are listed in Table I. The sequence of the control templates was a mock sequence that did not specifically bind to any mRNA from the mouse genome. DNA templates against four different DNA coding (sequence coding for the amino acids in a protein, CDS) regions of Spastin siRNA were designed online through BLOCK-iT™ RNAi Designer (http://rnaidesigner.invitrogen.com/rnaiexpress/) with some modifications. The sequence specificity was verified through a BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) homology search.
siRNAs were produced using the T7 RiboMAX™ Express RNAi System (Promega Corporation), according to the manufacturer’s instructions. Briefly, for each double-stranded siRNA against one of the four Spastin CDS regions, two pairs of synthesized complementary single-stranded DNA oligonucleotides were first annealed to form two double-stranded DNA templates. Subsequently, two complementary single-stranded siRNAs were separately synthesized in accordance with these two templates and then annealed to form a final double-stranded siRNA. Next, the siRNA was purified by conventional phenol/chloroform/isopropanol precipitation and then aliquoted and stored at -80°C after a quality check on an agarose gel. A ready-to-use siRNA mixture was prepared by mixing the siRNAs against four target regions together at an equal molar ratio to a final concentration of 5 µM.
Microinjection of siRNA into the cytoplasm of fully-grown immature oocytes was used to knock down Spastin. After injections, oocytes were arrested at GV stage with 2.5 μM milrinone for 20 hours, and then were cultured in milrinone-free M2 medium for maturation.
Immunofluorescence. The oocytes were briefly washed in PBS with 0.05% polyvinylpyrrolidone (PVP), permeated in 0.5% Triton X-100/PHEM (60 mM PIPES, 25 mM HEPES pH 6.9, 10 mM EGTA, 8 mM MgSO4) for 5 min and washed three times rapidly in PBS/PVP. Next, the oocytes were fixed in 3.7% paraformaldehyde (PFA)/PHEM for 20 min at room temperature, washed three times (10 min each) in PBS/PVP and blocked with blocking buffer (1% BSA/PHEM with 100 mM glycine) at room temperature for 1 h. Then, the oocytes were in sequence incubated at 4°C overnight with a primary antibody diluted in blocking buffer, washed three times (10 min each) in PBS with 0.05% Tween-20 (PBST), incubated at room temperature for 45 min with a secondary antibody diluted in blocking buffer (1:750 in all cases), and washed three times (10 min each) in PBST. Finally, the DNA was stained with 10 µg/ml Hoechst 33258 (Sigma-Aldrich; Merck KGaA) at room temperature for 10 min, and the oocytes were mounted onto a slide with mounting medium (0.5% propyl gallate, 0.1 M Tris-HCl, pH 7.4, 88% glycerol) and covered with a cover glass (thickness, 0.13-0.17 µm). To maintain the dimension of the oocytes, two strips of double-stick tape (thickness, 90 µm) were placed between the slide and cover glass. The primary antibodies were diluted as follows: anti-Spastin, 1:200; anti-tubulin, 1:500; anti-human kinetochore, 1:500. The oocytes were examined with an Andor Revolution spinning disk confocal workstation (Oxford instruments).
Western blotting. A total of 100 oocytes were lysed in Laemmli sample buffer (Bio-Rad Laboratories, Inc.) containing a protease inhibitor and boiled for 5 min before being subjected to 10% SDS-PAGE. The separated proteins were transferred to a PVDF membrane and then blocked in TBST (TBS containing 0.05% Tween-20) with 5% nonfat milk at room temperature for 1 h. Then, the PVDF membrane was separated and incubated overnight at 4°C with primary antibodies as follows: mouse monoclonal anti-β-actin (cat. no. A5316-100) was diluted with a blocking buffer (TBS containing 0.05% Tween-20) at a ratio of 1:1,000; Mouse monoclonal anti-SPASTIN (A-4) (cat. no. sc-398264) was diluted with a blocking buffer at a ratio of 1:500 was diluted with a blocking buffer at a ratio of 1:2,000. After being washed in TBST, the membranes were incubated with HRP-conjugated rabbit anti-goat IgG or HRP-conjugated goat anti-mouse IgG (diluted with a blocking buffer to 1:1,000) for 1 h at room temperature and then processed using an ECL Plus Western Blotting Detection System (Vazyme). In the experiment, we used imajeJ 1.8.0 as data analysis software.
Data analysis and statistics. All experiments were repeated at least three times. Measurements on confocal images was performed with ImageJ (National Institutes of Health). Data are presented as the average ± sem. Statistical comparisons were performed with Student’s t-test in Excel. P<0.05 was considered to indicate a statistically significant difference.