B6D2F1 (DBA/2 × C57BL/6) male/female mice were purchased at 7–10 weeks in age from Beijing Vital River Laboratory Animal Technology Co., Ltd, Beijing, China. The mice were kept in clean cages, and the surrounding temperature was maintained between 17 ℃ and 22 ℃. The lights were turned on from 5:00 to 19:00. All animal experiments were implemented in line with the Guidelines for Animal experiments of the Harbin Medical University (HMUIRB20190011). The approaches were approved by Harbin Medical University Ethics Committee. Unless stated, all reagents for embryo culture were purchased from Sigma (USA).
Oocyte and zygote collection and spermatozoa preparation
Female B6D2F1 mice were superovulated by injecting 5IU pregnant mare serum gonadotropin (PMSG, NSH, China), followed by 5IU human chorionic gonadotropin (hCG, NSH, China) 48 h later. To obtain zygotes, female mice were mated with male B6D2F1 mice after hCG injection. Approximately 14 h and 17 h after HCG injection, the oocytes and zygotes were collected from the fallopian tubes. Mice were anesthetized by 2,2,2-tribromoethanol (125mg/kg), and then euthanized by carbon dioxide suffocation after collection. Corpse were frozen for uniform disposal. Cumulus-free oocytes with appropriately sized perivitelline space and homogeneous ooplasm were selected, while only zygotes with two distinct parental pronuclei were selected. They were placed in 20 μl-droplets of CZBG, and covered by sterile mineral oil (Fisher, O121–20). Then, the oocytes were placed in an incubator set to 37℃ and 5% CO2 until use. Spermatozoa were collected from the cauda epididymis of male B6D2F1 mice at 8–10 weeks in age. The spermatozoa were then placed in CZB-HEPES medium until injection.
Generation of ICSI, PT, and DS embryos
ICSI, implemented by a piezo-driven unit following a previously described method , was treated as control; however, our experiments were performed in HEPES-CZB containing 5 μg/ml cytochalasin B (Sigma, C6762) at room temperature. The ICSI-generated embryos were washed at least three times, and were placed in KSOM in an incubator set to 37℃ and 5% CO2, after the sperm head was injected into the oocyte. PT androgenetic embryos were constructed following a previously described method ; however, our study used different electroporation parameters. We used two direct-current-pulses of 1.8KV/cm for 10 μsec each to complete cytoplast-karyoplasm fusion. In addition, DS androgenetic embryos were constructed following previously described methods [14, 23].
Immunofluorescent staining and image analyses
PT and DSandrogenetic embryos, as well as parthenogenetic embryos constructed by the maternal pronucleus, were transferred at 10 h post fertilization. The zona pellucida was dissolved using acidic CZB-HEPES solution. All samples were fixed in 4% paraformaldehyde (Sigma, PFA, P6148) in phosphate-buffered saline (PBS) at room temperature for 40 min. The samples were then permeabilized in 0.2% Triton X–100 in PBS at room temperature for 1 h, after washing several times in HEPES-CZB. To observe 5mC and 5hmC, permeabilized embryos were additionally placed in 2N HCL solution at room temperature for 50 min. The embryos were blocked with 1% bovine serum albumin (BSA) and 0.2% Triton X–100 in PBS overnight at 4 °C. Then, the embryos were incubated with anti-Tet3 (monoclonal; Proteintech), anti–5hmC (polyclonal; Active Motif), and anti–5mC (monoclonal; Calbiochem) overnight at 4 °C. The embryos were washed several times in 0.01% Tween 20 in PBS (PBST). They were then transferred to a secondary antibody mixture of Alexa Fluor 488 (anti-rabbit, Invitrogen) and Alexa Fluor 568 (anti-mouse, Invitrogen) at room temperature for 1 h in a dark place. After the nuclei were stained with 10 μg/ml Hoechst 33342, the embryos were mounted on slides with anti-fluorescence-fade medium (DABCO), and were examined by a laser-scanning confocal microscope (Zeiss, LSM700). In addition, we collected PT and DS blastocysts at 108 h post fertilization to examine the number of cells in the inner cell mass (ICM) and trophectoderm (TE) cells. Anti-OCT4 (monoclonal; Santa Cruz Biotechnology) for ICM and anti-CDX2 (monoclonal; Proteintech) for TE were used in this study. Tet3, 5mC, and 5hmC dots were quantitatively analyzed with Image-pro plus version6.3.
To examine H3.3 protein expression in different androgenetic embryos, the male pronucleus from zygotes and double sperm heads-formed male pronucleus were collected. The female pronucleus from zygotes was treated as the control. We selected 10 h post fertilization as the examined time point. We used maternal pronucleus transfer (MT) embryos as the negative control. The same amounts of pronuclei were collected in each group. The number of pronuclei served as a reference in this experiment, rather than the Housekeeping gene. Twenty pronuclei from each group were placed in SDS sample buffer (10 mmol/L, pH 6.8, Tris-HCL, 20 mmol/L, 4% SDS, 0.2% bromophenol blue, and 20% glycerol), and heated to 95 °C for 5 min. Details of the procedure are provided in Zhao et al. . The anti-H3.3 primary antibody (Rabbit polyclonal; Abcam) and the horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA) were used for this study. H3.3 signals were observed by the DAB detection system.
Reverse transcription and quantitative PCR analysis
The PT, DS, and ICSI embryos were collected at 78, 96, 108 h post fertilization as morulae, early blastocysts, and late blastocysts, respectively. mRNA from pools of 10 embryos was extracted using the Dynabeads mRNA DIRECT Micro Kit (Invitrogen, 61021), and were then used for reverse transcription with the High Capacity cDNA Reverse Transcription Kit (ABI, 4368814), following the manufacturer’s instructions. Quantitative PCR (qPCR) was implemented using Top Green qPCR SuperMix (Transgen, AQ131) on a CFX96 Real-Time System (Bio-Rad, USA). The results were calculated using the 2△△Ct method. The expression of target cDNA was normalized to the expression of Gapdh. Primer sequences are listed in Table 1.
The general linear models (GLM) procedure in the Statistical Analysis System (SAS User’s Guide, 1985, Statistical Analysis System Inc., Cary, NC) was used to analyze the data from all the experiments. Significant differences were determined using Tukey’s multiple range test. P < 0.05 was considered significant.