Mice
Mice were housed in constant temperature at 23 °C ± 1 °C with 40% humidity under a 12 h light–dark cycle. Eight-week-old female B6D2F1 [BDF1, cross between C57BL/6J (B6) female x DBA/2J (D2) male] mice were purchased from Orient Bio (Gyunggi-do, Korea). Mice between 10‒14-weeks-old were used in the “young” group, whereas mice older than 45 weeks were used in the “old” group. All experiments were conducted in accordance with the policies of the Konkuk University Institutional Animal Care and Use Committee (Approval number KU17067). Mice were sacrificed under anesthesia, and all efforts were made to minimize suffering of the mice.
Oocyte collection
Mice received 10 IU of pregnant mare’s serum gonadotropin (PMSG, Sigma-Aldrich, St. Louis, USA) intraperitoneally, and 10 IU of human chorionic gonadotropin (hCG, Sigma-Aldrich) 48 h later. Ovulated cumulus-oocyte complex (COCs) were retrieved from the oviduct 13 h post-hCG. 20 IU of PMSG and hCG injections were used for better induction in older mice, as in previously conducted research [10,23]. To remove the cumulus cells, the COCs were treated with hyaluronidase (300 mg/ml, H4272, Sigma Aldrich) in Quinn’s Advantage Medium with HEPES (SAGE Media, ART-1023, Trumbull, CT, USA) for 2 min at room temperature. Denuded metaphase II (MII) oocytes were washed and collected in Quinn’s Advantage Medium with HEPES containing 20% fetal bovine serum (FBS, Gibco; Grand Island, NY, USA).
Vitrification and warming
Vitrification was performed as previously described by Cha et al. [24]. Ethylene glycol (EG, 102466, Sigma-Aldrich) and dimethyl sulfoxide (DMSO, D2650, Sigma-Aldrich) were used as cryoprotectants in the vitrification solution. Oocytes were equilibrated in PBS based media containing 7.5% EG, 7.5% DMSO, and 20% FBS for 2.5 min, and then transferred to media containing 15% EG, 15% DMSO, and 0.5 M sucrose (Fisher Scientific, Fair Lawn, USA) for 20 sec. Equilibrated oocytes (20 to 25) were loaded onto a copper grid (Ted Pella Inc, Redding, USA) and dipped directly into liquid nitrogen (LN2). Vitrified oocytes were stored in a LN2 tank for 2‒4 weeks. For the warming procedure, the grid was taken out from the LN2 tank and serially incubated in 20% FBS containing PBS with descending concentrations of sucrose (0.5, 0.25, 0.125, 0 M) for 2.5 min each. The vitrified-warmed oocytes were washed in Quinn’s Advantage medium containing HEPES and 20% FBS. Washed oocytes were cultured in M16 media (M7292, Sigma-Aldrich) at 37 °C, in 5% CO2 for 1 or 3 h. For Necrostatin-1 (Nec1, N9037, Sigma-Aldrich) supplementation, 1 μM of Nec1 [25] was added to the final vitrification solution (15% EG, 15% DMSO, and 0.5 M sucrose). Oocytes without any marked morphological deformation and discoloration under an inverted microscope were considered as survived ones and used for further analysis.
RNA extraction and quantitative real-time polymerase chain reaction analysis
MII oocytes obtained from multiple mice were pooled and randomly grouped in 20 for RNA extraction. mRNA was extracted from 20 oocytes in all experimental groups, using DynabeadsTM mRNA DIRECTTM Purification Kit (Life Technologies, Forster City, CA, USA) according to the manufacturer’s protocol. The mRNA was kept in -80 °C before use. First strand cDNA was synthesized from total mRNA sample using a Superscript TM Ⅲ Reverse Transcriptase (Invitrogen, 18080-044, Carlsbad, CA, USA), RNaseOUTTM Recombinant Ribonuclease Inhibitor (Invitrogen, 10777-019), Oligo (dT)20, and random hexamer primers (Roche, Basel, Switzerland). Real-time quantitative PCR (qPCR) was performed using 2 μl of oocyte cDNA (equivalent to one oocyte per reaction) and Applied Biosystems™ Power Up™ SYBR™ Green Master Mix (Invitrogen, A25742, Carlsbad, CA, USA) in a final volume of 20 μl on the ABI 7500 real-time PCR system. The PCR conditions were as follows: hold for 10 min at 95 °C, followed by each cycle consisting of denaturation at 95 °C for 15 sec, annealing and elongation at 58 °C for 1 min each. The relative gene expression was normalized with H2afz mRNA expression and relative quantification was performed using the ddCt method [26,27]. PCR was performed by using Econo Taq PLUS GREEN 2X Master Mix (Lucigen, Middleton, WI, USA). Three biological replicates were used per experimental group and all reactions were run in duplicates. Primers used are shown in Table 1.
Cell culture and necroptosis induction
L929 fibroblast cell line derived from mouse adipose tissue was obtained from Korean Cell Line Bank (Seoul, Korea). L929 cells were cultured in RPMI1640 media supplemented with L-glutamine (300 mg/L), 25 mM HEPES, 25 mM NaHCO3, and 10% FBS. To induce necroptosis, cells were treated with a mixture of 30 ng/mL TNFα (PeproTech, Rocky Hill, NJ), 10 μM LCL-161 (R&D Systems, Minneapolis, MN, USA), and 20 μM Z-VAD-FMK (R&D Systems) for 40 min [28]. Cells were fixed and subjected to immunofluorescence staining with anti-pMLKL and anti-pRIPK1 antibodies to establish specificity of these antibodies.
Immunofluorescence staining
Oocytes were fixed with 4% paraformaldehyde containing 0.05% polyvinyl alcohol in phosphate buffered saline (PFA-PVA) for 10 min. Fixed oocytes were washed three times with phosphate-buffered saline containing 0.05% polyvinyl alcohol (PBS-PVA) for 10 min each. For permeabilization, oocytes were transferred to a solution containing 0.25% Triton X-100 and incubated for 10 min. To prevent nonspecific binding, oocytes were blocked with 2% BSA in PBS for 1 h, followed by incubation with primary antibody at 4 °C overnight. The primary antibodies used were anti-pMLKL (1:100, ab196436, Abcam) [21], anti-pericentrin (1:500, 611814, BD Bioscience, San Jose, CA, USA) [29], and anti-pRIPK1 (1:150, 31122, Cell Signaling Technology, Danvers, MA, USA). Following incubation with primary antibodies, oocytes were washed three times with PVA-PBS for 10 min. The oocytes were then incubated with Alexa Fluor 488 or Alexa Fluor 568 conjugated secondary antibody (1:250, Invitrogen) for 1 h at room temperature. DNA was stained with TOPRO-3-iodide (1:250, Invitrogen). The oocytes were mounted on glass slides with Vectashield mounting medium (Vector Laboratories, Peterborough, UK) and observed under the confocal microscope (Zeiss LSM710, Carl Zeiss AG, Oberkochen, Germany). Specificity of anti-pMLKL and anti-pRIPK1 antibodies was confirmed in necroptosis-induced L929 cells [21]. In all experiments, rabbit IgG was used as a negative control and it did not generate any specific signal.
Live imaging of oocytes by confocal microscopy
Oocytes were washed with M16 media three times and stained with CellMaskTM Plasma Membrane Stain (2.5 μg/ml; C10046, Life technologies), BODIPY 500/510 dodecanoic acid (10 μg/ml; D-3823, Invitrogen), or ER TrackerTM Red dye (1 μg/ml; E34250, Invitrogen) for 30 min. The oocytes were rinsed with fresh M16 media three times and transferred to a glass bottom confocal dish. Live images of oocytes were obtained directly with a confocal microscope (Zeiss LSM710).
Statistical analysis
Data analysis and graph preparation were done using GraphPad Prism 5 software. For statistical analysis, Student’s t-test or one-way analysis of variance (ANOVA) were conducted on the experimental groups. Tukey’s range test was then performed to identify whether a significant difference exists among the groups. Statistical significance was depicted as *: p <0.05, **: p<0.01 and ***: p<0.001.