The oligonucleotides used herein were synthesized by Genotech (Daejon, Korea). The restriction enzymes and the DNA ligation kit were purchased from Takara (Tokyo, Japan). The QIAprep-Spin plasmid kit was acquired from QIAGEN, Inc. (Hilden, Germany). The Lumi-Light western blot kit was purchased from Roche (Basel, Switzerland), and the pcDNA3 mammalian expression vector, FreeStyle CHO-S suspension cells, PNGase F, FreeStyle MAX transfection reagent, and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, USA). The PMSG ELISA kit was purchased from DRG International, Inc. (Mountain side, NJ, USA), Centriplus Centrifugal Filter Devices from Amicon Bio separations (Merck, Billerica, MA, USA), and an anti-myc antibody and antibodies against HSD17b1, ADAMTS1, Edn2, and OVGP1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Disposable spinner flasks were obtained from Corning Inc. (Corning, NY, USA). A peroxidase-conjugated anti-mouse IgG antibody was obtained from Bio-Rad (Hercules, CA, USA), whereas pregnant-mare serum gonadotropin (eCG; ≥1,000 IU/mg, G4877) and hCG (5,000 IU, CG5) from Sigma-Aldrich Corp. (St. Louis, MO, USA), as were all other reagents. PMSG and hCG reagents are generally used to induce the ovulation in mice as we have previously reported . All protocols complied with the approved Guidelines for Animal Experiments of Hankyong National University, Korea, and were approved by the Animal Care and Use Committee of Hankyong National University, Korea (Approval ID: 2015-8).
Construction of tethered eCG gene
cDNA encoding the tethered R-eCGβ/α was inserted into the mammalian expression vector pcDNA3, as previously reported . The same method was used to insert a myc tag (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) between the first and second amino acid residues of the β-subunit of the mature eCG protein . Plasmid DNA was then purified and sequenced in both directions through automated DNA sequencing to ensure correct inserts. The cloned expression vector of tethered eCG was designated as pcDNA3-eCGb/a, as previously reported . A schematic representation for tethered R-eCG β/α is shown in Fig. 1.
Cell culture and generation of tethered R-eCG
In CHO-S cells, the tethered R-eCG expression vector was transfected into CHO-S cells using the FreeStyle MAX reagent (Invitrogen; Carlsbad, CA, USA) transfection method, in accordance with manufacturer's instructions. Flasks were placed on an orbital shaking platform, rotating at 120–135 rpm at 37°C in a humidified atmosphere of 8% CO2 in air. On transfection, the cell density was approximately 1.2–1.5 × 106 cells/mL. The plasmid DNA (260 ug) and a FreeStyleTM MAX Reagent complexes were gradually added to 200 mL of medium containing cells. Finally, culture media were sampled on day 9 after transfection and centrifuged to eliminate cell debris. The supernatant was sampled and stored at -20°C until the assay. The samples were concentrated using a Centricon filter or by freeze-drying and mixed with PBS.
Quantification of R-eCG proteins
R-eCG protein was quantified with the PMSG ELISA kit (DRG Diagnostics; Mountain side, NJ, USA). Briefly, the PMSG standard and R-eCG samples (100 µL) were dispensed into the wells of a plate coated with the antibody and incubated for 60 min at ambient temperature. After rinsing thrice, 100 µL of anti-PMSG antibody conjugated with horseradish peroxidase was added into each well and incubated for 60 min. The plate wells were rinsed five times, and substrate solution (100 µL) was added and incubated for 30 min at ambient temperature. Finally, 50 µL of a stop solution was added and the absorbance was measured at 450 nm, using a microtiter plate reader CytationTM 3 (BioTeK, Winooski, VT, USA). The average absorbance of each standard was plotted against its corresponding concentration in a linear–log graph. We determined the average absorbance of each sample to determine the corresponding PMSG value via simple interpolation through a standard curve. Given the low expression level of R-eCG in CHO-S cells, samples were concentrated approximately 40 ~ 50 times for application of R-eCG in MCR and superovulation. Concentrated R-eCG samples were diluted about 40 times for standard curve calibration. Samples for the standard curve were 0. 25, 100, 200, 400, 800 mIU/mL. Finally, 1 IU was considered 100 ng in accordance with the conversion factor of the suggested assay protocol.
Detection of R-eCGs via western blotting and enzymatic digestion of N-linked oligosaccharides
Concentrated sample media were subjected to SDS-PAGE (12.5% resolving gel) via the Laemmli method . After SDS-PAGE, the proteins were electro-transferred to a nitrocellulose membrane for 2 h in a Mini Trans-Blot Electrophoretic Transfer cell. To eliminate all N-linked oligosaccharides, the R-eCG sample was incubated for 24 h at 37°C with PNGase F [2 mL of the enzyme (2.5 U/mL) per 30 mL of sample+8 mL of 5´ reaction buffer]. The reaction was terminated by boiling for 10 min, and the samples were subjected to SDS-PAGE and the proteins were electro-transferred on to a membrane. After blocking the membrane with a 1% blocking reagent for 1 h, followed by probing with monoclonal anti-myc antibody (1: 5,000) for 2 h, the membrane was washed and probed with a secondary antibody (peroxidase-conjugated anti-mouse IgG antibody 37.5 μL/15 mL of the blocking solution) for 30 min. The membrane was then incubated for 5 min with 2 mL of the Lumi-Light substrate solution and X- ray film was exposed to the membrane for 1–10 min.
Assessment of the MCR of N-eCG and R-eCG
Each animal was intravenously administered 5 IU of N-eCG or R-eCG through the tail vein to determine the 50% dose for the induction of superovulation. Blood was sampled from the transorbital vein in heparinized microhematocrit tubes. Blood samples were obtained at 10 and 30 min and at 1, 2, and 24 h and centrifuged for 15 min at 5,000 rpm at 4°C, and plasma eCG concentrations were estimated using the PMSG ELISA kit (DRG Diagnostics).
The MCRs of N-eCG and R-eCG were determined in 8-week-old male B6D2F1 (C57BL6 ´ DBA/2) 12 mice. The female 16 mice (8-week-old B6D2F1; Oriental Bio, Gyeonggi, Korea) were superovulated by injection of 10 IU of N-eCG and R-eCG and then 10 IU hCG after 48 h. The ovarian tissues were sampled at 13 h after hCG administration. All mice were euthanized with carbon dioxide inhalation, and the ovarian tissues were collected at the end of study. All the mice were raised in an environment with the temperature of 23 ± 1 °C with regular 12 h light/dark cycle and allowed free access to feed and water. The animals were processed according to the Animal Care and Use Committee procedure. The protocol was approved by the Committee on Ethics of Animal Experiments at the Hankyong National University (Approval ID: 2015-8).
Total RNA was extracted from ovaries, using TRIzol reagent, and purified using RNeasy columns in accordance with the manufacturers’ protocols, as previously described .
1) Labeling and purification
Total RNA was amplified and purified using an Ambion Illumina RNA amplification kit (Ambion, Austin, TX, USA) in accordance with the manufacturer's instructions to obtain biotinylated cRNA. Briefly, 550 ng of total RNA was reverse-transcribed into cDNA with a T7 oligo(dT) primer. Second-strand cDNA was synthesized, transcribed in vitro, and labeled with biotin-NTP.
2) Hybridization and data export
Labeled cRNA samples (0.75 µg) were hybridized to the Illumina MouseRef-8 v2 expression BeadChip (Illumina, Inc., San Diego, CA, USA) for 16–18 h at 58°C. Array signals were detected using Amersham Fluorolink Streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) in accordance with the manufacturer's instructions. Arrays were scanned using an Illumina bead array reader (confocal scanner). Array data were analyzed using in Illumina Genome Studio v.2009.2 software (Gene Expression Module v.1.5.4).
3) Raw data preparation and statistical analysis
Raw data were extracted using the software provided by the manufacturer (Illumina Genome Studio v.2009.2) and filtered using a detection p-value of <0.05 (a signal value higher than that of the background was necessary to set the detection p-value of <0.05). The selected gene signal value was logarithmically transformed and normalized to XYZ. Comparative analysis between two groups was conducted on the basis of the p-value evaluation, via the local-pooled-error test (adjusted Benjamini–Hochberg false discovery rate had to be<5%) and the fold-change. Biological ontology-based analysis was performed for the Panther database (http://www.pantherdb.org). Furthermore, genes whose expression levels differed by >2-fold were considered differentially expressed between the two groups.
RT-PCR and qRT-PCR analyses
To validate the microarray data, 14 genes (up-regulated: Tex19.2, Sectm1b, Ctsk, Gpnmb, Sectm1a, and Hsd17b1; down-regulated: OVGP1, BC048546, Tmem68, Dcpp1, Prkg2, Edn2, Adamts1, and Akr1b7) from different groups were evaluated through RT-PCR and qRT-PCR analysis, their expression levels differed by >2-fold. RT-PCR and qRT-PCR analysis was performed for the same ovarian tissue subjected to microarray analyses. Primer sequences are outlined in Supplementary Material Table 1 along with the primer annealing temperatures. The primers were designed using Primer3 software (http://www.bioneer.co.kr/tools/). Ovarian gene expression levels were then normalized to those of Actb via the 2-ΔΔCT method for quantitative relation.
Immunohistochemical staining of ovarian samples was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) in accordance with the manufacturer's instructions. The samples were fixed in 10% neutral-buffered formalin at ambient temperature for 24 h and washed with PBS. Thereafter, the fixed samples were rehydrated in graded ethanol (EtOH) solutions (3 min each in 100% 2´; 95% 1´; 70% 1´; and 50% 1´) and embedded in paraffin. Paraffin-embedded tissues were sectioned into 8-µm-thick sections, which were then mounted onto poly-l-lysine-coated slides. The slides were boiled in 10 mM sodium citrate for 10 min and chilled on ice for 20 min. Thereafter, they were washed with 3% hydrogen peroxide for 10 min and blocked for 1 h at ambient temperature. The slides were incubated with the primary antibody and then with an anti-rabbit IgG antibody (secondary antibody). Finally, the slides were immunostained using the ABC detection kit in accordance with the manufacturer's instructions and stained with DAB. The slides were examined under a Nikon Eclipse TE-2000-E confocal microscope (Tokyo, Japan).
Data and statistical analysis
Data are presented as mean ± SEM values. One-way ANOVA with Tukey’s multiple-comparison test was conducted to compare the results between samples. In figures, the superscripts indicate significant differences between groups (p< 0.05).