In vitro embryo production Mature oocytes were obtained using the method described in our previous study [35]. Briefly, prepubertal gilt ovaries collected in Anseong, Kyunggi Province, Korea, were used in this experiment. The follicular fluid and cumulus-oocyte complexes (COCs) were aspirated using an 18-gauge needle and then pooled to obtain sediments. The sediments were washed with TL–HEPES–PVA medium, and the oocytes with compact cumulus cells and granulated cytoplasm were selected for in vitro maturation. The washed COCs were cultured in tissue culture medium (TCM-199; Life Technologies, Carlsbad, CA, USA) containing 10 ng/ml epidermal growth factor, 1 mg/ml insulin, and 10% porcine follicular fluid for 44 h at 39.8°C at 5% CO2 and 100% humidity. The COCs were treated with a 4 IU/ml solution of the hormones Q6 equine chorionic gonadotropin and human chorionic gonadotropin (Intervet, Cambridge, UK) for the first 22 h. The COCs were then matured under hormone-free conditions. To generate parthenotes, the cumulus-free oocytes were activated with an electric pulse (1.0 kV/cm for 60 ms) in activation medium (280 mM mannitol, 0.01 mM CaCl2, 0.05 mM MgCl2) using a BTX Electro-cell Manipulator (BTX, CA, USA), followed by 4 h of incubation in porcine zygote medium-3 (PZM-3) medium containing 2 mmol/l 6-dimethylaminopurine. Subsequently, the zygotes were transferred in groups of 25–50 to wells with 500 μl PZM-3 medium for 7 days with or without linoleic acid (L-9530) or APDC (P8765) from Sigma.
Immunocytochemistry
Embryos without zona pellucida at each stage were fixed in 4% paraformaldehyde for 30 min at 4°C. The fixed samples were permeabilized using 1% Triton X-100 for 5 min at room temperature and washed three times with phosphate-buffered saline (PBS). The embryos were blocked using 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature, and primary antibodies targeting NF-kb p65 (SC-8008, mouse IgG; Santa Cruz), IL-6 (AHP-2391, rabbit IgG; Bio-Rad) and C-JUN (PA5-17890, rabbit IgG; Invitrogen) were added overnight at 4°C. The primary antibodies were diluted 1:500 in PBS containing 1% BSA. The embryos were washed three times with PBS containing 0.1% Tween-20 before incubation with the fluorescent-conjugated secondary antibodies anti-mouse IgG (green, 1:500, A11008; Invitrogen, Carlsbad, CA, USA) and anti-rabbit IgG (red, 1:500, A11012; Invitrogen), which were diluted in a blocking solution, for 2 h at room temperature. The samples were washed three times with PBS containing 0.1% Tween-20, and the nuclei were stained using 0.1% Hoechst 33342 (Molecular Probes, Eugene, OR, USA) for 10 min. After washing three times with PBS, the samples were mounted on a slide glass. The stained samples were visualized under a microscope (Eclipse TE2000-U; Nikon), and the captured images were processed using a Nikon digital sight DS-L1.
Measurement of apoptosis
Terminal deoxynucleotidyltransferase (TdT)-mediated deoxy-uridine nick-end labeling (TUNEL) is the enzymatic addition of fluorescently labeled nucleotides to the free 3′-ends of DNA strands made available by DNA fragmentation that typically accompanies programmed cell death or apoptosis. To visualize apoptotic nuclei, the embryos were washed four times at room temperature in phosphate buffered saline (PBS) supplemented with 1 mg polyvinyl pyrrolidone (PVP) per milliliter PBS (PBS-PVP). The embryos were then fixed for 1 h at room temperature in 4% (w/v) paraformaldehyde in PBS (pH 7.4). After fixation, the embryos were washed an additional two times in PBS-PVP. The embryos were then permeabilized for 10 min at room temperature in a 0.5% (v/v) Triton X-100 solution in PBS. After permeabilization, the embryos were washed one time in PBS-PVP in preparation for the TUNEL procedure. The fixed and permeabilized embryos were then subjected to the TUNEL assay procedure using an in situ cell death detection kit (Roche; Mannheim, Germany) according to the manufacturer’s instructions. Briefly, the embryos were incubated with terminal transferase enzyme and labeled nucleotide solution (mixed in a 1:10 ratio) in a humidified, sealed chamber in the dark at 37°C for 1 h. After completion of the TUNEL reaction procedure, the embryos were washed three times for 5 min in PBS-PVP and then transferred to a solution containing 10 mg/ml Hoechst 33342. Nuclear staining by 0.1% Hoechst 33342 was allowed to proceed for approximately 10 min at room temperature in the dark. The nuclei displaying distinct labeling and condensed or pyknotic morphology were considered to be TUNEL-positive. The average embryo cell numbers were determined by counting the number of nuclei stained with the fluorescent nuclear dye Hoechst 33342.
Quantitative RT-PCR
Pooled embryos, each stage of the in vitro-produced embryos (2–3-cell, n = 20; 4-cell, n = 20; 6–8-cell, n = 20; morula, n = 10; and BL, n = 5), and single blastocysts were processed with a Dynabeads® mRNA DIRECT™ Kit (Invitrogen) following the manufacturer’s instructions. The zona pellucida was removed using Tyrode’s acid before mRNA extraction. cDNA was synthesized using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA, USA). The extracted cDNA samples were amplified using a DyNAmo HS SYBR Green qPCR Kit (Thermo Fisher Scientific, MA, USA) containing 1–2 pmol of each primer set listed in Table 1 in a 10-µl reaction volume. The amplification and detection were conducted using the ABI 7300 Real-Time PCR system (Applied Biosystems) under the following conditions: one cycle of 50°C for 2 min and 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension for 1 min (the annealing/extension temperatures were dependent on each primer set). The dissociation curves were analyzed, and the amplified products were loaded onto gels to confirm the specificity of the PCR products. The relative expression levels were calculated by normalizing the threshold cycle (Ct) values of each gene to that of the reference gene GAPDH via the delta-delta Ct method.
Table 1: Primer sequences used for real-time PCR.
Genes
|
Sequences
|
Size
|
RelA
|
5'- CGGGGACTACGACCTGAATG -3'
|
158
|
5'- CTTTCTGCACCTTGTCGCAC-3'
|
C-JUN
|
5'- AGCAGAGCATGACCCTGAAC-3'
|
132
|
5'- ACTGGATTATCAGGCGCTCG-3'
|
C-FOS
|
5'- CAAGCGGAGACAGACCAACT-3'
|
105
|
5'- GTGAGCTGCCAGGATGAACT-3'
|
BAK1
|
5'- CAGCCGACAGCGGAAAAC-3'
|
109
|
5'- GGTAGCCAAAGCCCAGAAGA-3'
|
TP53
|
5'- CCCATCCTCACCATCATCAC-3'
|
80
|
5'- GCACAAACACGCACCTCAA-3'
|
CASP3
|
5'- GAACTCTAACTGGCAAACCCAA-3'
|
84
|
5'- CACTGTCCGTCTCAATCCCA-3'
|
BCL-XL
|
5'- ACTGTGCGTGGAGAGCGTAG-3'
|
87
|
5'- AGGTGGTCATTCAGGTAAGTGG-3'
|
MCL1
|
5'- GTTTGGCCTCCAGAGAAACG-3'
|
120
|
5'- TTTCCCGTAGCCAAGAGACG-3'
|
IL-6
|
5'- GCAGTCACAGAACGAGTGGA-3'
|
146
|
5'- CTCAGGCTGAACTGCAGGAA-3'
|
ACTB
|
5’-GTGGACATCAGGAAGGACCTCTA-3’
|
131
|
5'- ATGATCTTGATCTTCATGGTGCT -3'
|
Statistical analysis
The data obtained in this study were analyzed using the GraphPad Prism statistical program (GraphPad Software, San Diego, CA, USA). The data on the developmental rates were arcsine-transformed and then analyzed using analysis of variance and the Newman–Keuls multiple comparison test. The relative transcription levels in the embryos were analyzed using the unpaired Student's t-test. All the data are expressed as the means ± standard error of the mean. A probability of P < 0.05 was considered statistically significant.