As shown in Fig. S2, the control and MTX model mice were established by treating with normal saline and 5 mg/kg MTX. (The establishment of the effective dosage of MTX referred to Table S1.) This work studied the in-vivo matured oocytes. After retrieving the in-vivo matured oocytes from the control and MTX model mice, the in-vitro fertilization ability and the resulting embryo formation were firstly analyzed to confirm the effects of MTX on the oocyte quality. Then, the action mechanisms of MTX impairing oocytes quality were studied. From morphological point, fluorescence staining and imaging were used to examine the chromosome morphology, DNMT localization and spindle morphology. From the point of molecular biology, real-time PCR was used to examine the transcripts for genes of the key enzymes in the folate pathway, DNMTs, as well as those in the spindle assembly checkpoint and chromosome segregation, MAD2 and Sgo1. Additionally, we quantitatively measured the global DNA methylation using fluorescence polarization and measured the concentrations of SAM and SAH in the blood using high performance liquid chromatography (HPLC). Through the research above, we analyzed the effects of MTX on the in-vivo matured oocyte quality and its possible mechanisms.
The ICR mice (Chinese Academy of Medical Sciences, Beijing, China) were housed and bred in barrier environment. Food and water were available ad libitum according to the Chinese National Standard (GB 14925-2001).
24 hours after the treatment with MTX or normal saline, the model mice were treated with 10 IU pregnant mare serum gonadotropin (Bo’ en Pharmaceutical Ltd., Chifeng, China), and after 48h, were treated with 10 IU human chorionic gonadotropin (Livzon Pharmaceutical Group Inc., Zhuhai, China), then, were euthanized by cervical dislocation 15 h later. Cumulus-oocyte complexes (COCs) were harvested from the oviducts. Cumulus cells were isolated by brief incubation in 0.05% hyaluronidase (Sigma, Louis, MO, USA) at 37 °C for 5 min. This work selected the in-vivo matured oocytes for further study, and these selected oocytes had the features of arresting at the second meiotic metaphase, presenting morphology integrity and good refraction, showing the first polar body.
In-vitro fertilization, embryo culture and morphological examination
ICR male mice were euthanized by cervical dislocation, and their epididymal tails were removed and transferred to HTF droplets. Epididymal tails were punctured to slowly extrude sperm. Then, the sperm suspensions were capacitated in HTF droplets for 90 min. The capacitated sperms were added to the droplets containing COCs and cultured for 6 h. After washing, all the oocytes were transferred to KSOM (Millipore, Massachusetts, USA) droplets and cultured to the blastocyst stage for 4 days. The fertilization and embryo development were recorded by noninvasive imaging technology, full-field optical coherence tomography (FF-OCT), due to the advantages of label-free, three-dimension and high resolution. Fertilization was identified by the appearance of male and female pronuclei. Like our previous work (48), blastomere number was counted by the segmented nuclei which were the dark areas in the images captured by FF-OCT. Additionally, the blastomere localizations were used as a coarse retrieval to distinguish the inner cell mass (ICM) and trophoblast (48).
Immunofluorescence, two-photon fluorescence imaging and fluorescence intensity analysis
The in-vivo matured nude oocytes were fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS) for 30 min. Then, these fixed oocytes were permeabilized with 0.5% Triton-X 100 for 30 min, and were blocked in 2% normal rabbit serum blocking solution for 30 min. For DNMTs staining, they were incubated with anti-DNMT1, anti-DNMT3a, anti-DNMT3b and anti-DNMT3L (Jackson ImmunoResearch Laboratories, West Grove, USA) overnight at 4 °C in darkness. After washing, the oocytes were immersed in a biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, USA) for 30 min. Finally, these oocytes were rinsed and reacted with quantum dot 605-streptavidin conjugate (Invitrogen, California, USA) for 1 h. For spindle labeling, the blocked oocytes were incubated with FITC-conjugated anti-β-tubulin antibody (Sigma, Saint Louis, USA) in darkness for 1 h. Chromosomes were counterstained with Hoechst 33342 (2 μg/mL) for 15 min. The fluorescence images were captured by a Bio-Rad MRC 1024 system (Bio-Rad, California, USA) coupled to a Nikon TE300 inverted microscope (Nikon, Tokyo, Japan) with 100× oil objective (Plan Apochromat DIC H, NA 1.4; Nikon, Tokyo, Japan). All the groups experienced the same experimental conditions (namely, the same excitation laser power and wavelength, detector gain). We used software Amira 5.2 to assess the fluorescence intensity which was defined as the average grey value in the three-dimensional spatial region of interest (DNMTs). According to the method of Zhu (49), the fluorescence intensity in the control group was normalized to one, then, the relative fluorescence intensity in the MTX group was obtained.
Analysis of mRNA transcript levels
We used RNeasy Micro Kit (Qiagen, California, USA) to extract the total mRNA from about 35 oocytes. Then, RNA purity was estimated by ultraviolet-visible spectrophotometer and its integrity was detected by gel electrophoresis. Total RNA was reverse transcribed using Sensiscript RT Kit (Qiagen, California, USA). According to the manufacturer’s instructions, reverse transcription was performed using 20 μl reactions containing 2μl 10× buffer RT, 2μl dNTP Mix, 2μl Oligo-dT primer, 1μl RNase inhibitor, 1μl Sensiscript Reverse Transcriptase, 7μl RNase-free water, 7ng RNA template. Then, the 20 μl mixture was placed in a constant temperature bath at 37℃ for 60 min, and the cDNA was obtained. Each analysis was performed for three times using 15 μl reactions containing 7.5 μl SYBR Green PCR Master Mix (Applied Biosystems, UK), 0.6μl forward and reverse primers (10 μmol/L), 1.5μl cDNA, and 5.4 μl RNase free water. The sequences of the forward and reverse primers are shown in Table S2. The relative quantitation calculation was implemented by the 2-△△Ct method. β-actin was used as the internal reference.
Quantitative determination of global DNA methylation
The matured nude oocytes were immersed in Tyrode’s solution (Haling Biotechnology, Shanghai, China) to remove zona pellucid. After washing, these zona-free oocytes were immersed in the lysis buffer (Solarbio, Beijing, China) at 37℃ for 60 min. After sufficient dissociation, the mixed oocyte lysates were centrifuged 10000 rpm for 2 min, and then saved at -80℃.
According to the method of Zhao and Xue (50), a sample of 500 ng DNA was incubated at 37 ℃ for 12 h in a 50 μl reaction mixture containing 2.5 μl restriction enzyme HpaII (10 U/μg, TaKaRa Biotechnology, Dalian, China), 5 μl 10× Buffer T, 5 μl 0.1% BSA (Solarbio, Beijing, China), 500 ng DNA, and distilled water adding up to 50 μl. Another sample of 500 ng DNA was incubated at the same conditions, but HpaII in the reaction mixture was replaced by MspI (TaKaRa Biotechnology, Dalian, China). After enzyme digestion, 4 μl enzymatic products of HpaII/MspI were placed at 57.8 ℃ for 60 min in a 15 μl chain-extension reaction mixture containing 1.5 μl 10×PCR Buffer, 0.63 μl 0.75U Taq DNA polymerase (TaKaRa Biotechnology, Dalian, China), 0.56 μl TAMRA-dCTP (Applied Biosystems, UK), 4.0 μl restricted DNA and 8.3 μl distilled water. After TAMRA-dCMP incorporating into DNA, the chain-extension reaction products were measured by fluorescence polarization using microplate reader (Molecular SpectraMax Plus). The fluorescence polarization values of HpaII/MspI restriction products were recorded as FPHpaII and FPMspI. The global DNA methylation level of oocytes was calculated by (1- FPHpaII / FPMspI)×100%.
Quantitative measurement of SAM/SAH concentration in the eyeball blood
The concentration of SAM and SAH were measurement by extracting the mouse eyeball blood. According to the Zhen’s method (51), 100μl plasma sample were extracted with 10μl of 35% perchloric acid. Then, the acidified sample was vortexed for 1 min, subsequently, was placed at about 25℃ for 10min, lastly, were centrifuged with the speed of 15000 rpm at 4 ℃ for 10min. Ultimately, 25μl supernatant was analyzed using HPLC. HPLC equipment was Agilent 1100. The chromatogram column (Agilent ZORBAX C8, 250×4.6 mm, 5μm) was used for the analysis. The mobile phase consisted of two solvents: solvent 1 was 8 mM octane sulphonic acid and 50 mM NaH2PO4 (PH 3.0); solvent 2 was chromatographic grade methanol. The flow rate was 0.9 mL/min. The column temperature was 35 ℃. The UV detection wavelength was set at 450 nm from 0 to 8 min, and 254nm from 8 to 16 min. The SAM/SAH standards configuring in water to different concentrations were used to trace the radio-labelled peaks.
All the experiments were repeated three times. Results were presented as the means ± SEM. Statistical comparisons were performed by One-way ANOVA or chi-square using software SPSS 16.0. p<0.05 was considered significant.