The experimental protocol used in this study was based on an approved animal-use document and was in accordance to the guidelines of the Institute Animal Care and Use Committee (IACUC) of the National Institute of Animal Science (Approval No. 2017–219) at the Republic of Korea.

To isolate cOECs, an oviduct tissue from an egg-laying White Leghorn (WL) hen (30 to 35 weeks) was dissected, separated and dissociated, and the method used for the isolation of oviduct cells was modified from that used in a previous study [14]. In this study, 15-to-20 cm-long oviduct tissue from the infundibulum to the magnum was separated from the oviduct, and the isolated tissue was washed with 70% ethanol, washed twice with phosphate-buffered saline (PBS; Gibco, Carlsbad, CA, USA) supplemented with 1% antibiotic/antimycotic (anti-anti; Gibco, USA), and placed on the petri dish. The mesosalpinx of the tissue was trimmed, gently torn off using microsurgical scissors horizontally to expose the inner surface, and soaked with fresh PBS to avoid drying. The inner surface was then delicately scraped several times with surgical blade, and the scraped tissue fragments were finely minced into approximately 2-mm fragments with microsurgical scissors. The minced tissue fragments were centrifuged at 700 g and 4°C for 10 min, and the supernatant was removed. The pellets were digested with 10 mL (1 mg/mL) of collagenase P (Roche, Basel, Switzerland) supplemented with 10 mM HEPES (Sigma, Saint Louis, MO, USA) and placed in an incubator with 5% CO₂ at 37°C for 30 min, with vortexing every 5 min. The digested samples were centrifuged (1-step) at 700 g and 4°C for 10 min, and the supernatant was transferred with a fresh tube and centrifuged (2-step) again. The pellets from the first and second steps of the centrifugation were suspended in cell culture medium, and suspended tissue masses were totally placed on 100-mm collagen-treated dishes (Corning, New York, NY, USA) with Keratinocyte complete medium (K-SFM; Gibco, USA) supplemented with 5% chicken serum (Gibco, USA) and 1% anti-anti. The oviduct cells at passage 0 were isolated from the tissue masses in few days, observed under a microscope (Eclipse Ti, TE300; Nikon, Tokyo, Japan). Then, the cells from passage 1 were continuously cultured in K-SFM with 5% chicken serum, 1% anti-anti, and sub-cultured by trypsin treatment and centrifugation. The chicken embryonic fibroblast cells line DF-1 (CRL-12203; American Type Culture Collection, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 1% anti-anti, and 2% chicken serum.

The magnum portion of a chicken oviduct tissue was primarily fixed with 4% paraformaldehyde at 4°C, and washed three times with 0.05 M sodium cacodylate buffer. The sample was then secondarily fixed for 1.5 h with 1% osmium tetroxide in sodium cacodylate buffer, subjected to two brief washes, and stained overnight with 0.5% uranyl acetate at 4°C. For scanning and electron microscopy (SEM) analysis, the sample was dried twice with 100% isoamyl acetate for 15 min in a critical point dryer, mounted on metal stubs, coated with gold, and observed under a Bio-LV SEM (SN-3000; Hitachi, Tokyo, Japan).

The cOECs at passage 2 in a collagen-treated 75T flask were treated 200 μL of colcemid (Gibco, USA) stock solution, incubated at 37°C with 5% CO₂ for 4 h and harvested by centrifugation at 1,000 rpm for 10 min. After aspiration of the supernatant, the cells were suspended in 5 mL of hypotonic solution (0.075 M KCl) and incubated at 37°C for 10 min. Subsequently, 500 μL of Carnoy’s fixative (ethanol: acetic acid = 3:1) was added to the cells, and the cells were again harvested by centrifugation as described above. After aspiration, 3 mL of Carnoy’s fixative was added to cells, and the cells were incubated for at least 20 min and harvested by centrifugation at 1,000 rpm for 10 min, these steps were repeated. The pellets obtained after the final centrifugation were spread on a glass slide, and the slide was baked at 60°C for 30 min, and the chicken chromosomes were stained with Giemsa.

RNA from the oviduct magnum tissue (positive control), DF-1 (negative control), and cOECs at passage 2 was extracted using an RNA mini preparation kit (Qiagen, Hilden, Germany). For reverse transcription polymerase chain reaction (RT-PCR) analysis, total RNA (0.5 μg) was used for cDNA synthesis with the Superscript IV First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed with HS prime Taq DNA polymerase (Genet Bio, Daejeon, Korea) using SimpliAmp PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences (Table 1) were selected from the reference or designed with NCBI Primer Blast. Primer pairs were used to amplify specific region into chicken oviduct ovalbumin, ovomucoid, estrogen receptor 1 (ESR1), occludin, cytokeratin 14, E-cadherin, and β-actin genes. The PCR conditions consisted of 94°C for 10 min, followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, and 72°C for 5 min.

cOECs at passage 1 and DF-1 were seeded into a 4-well cell culture dish, fixed with 4% paraformaldehyde (Molecular probes, Eugene, OR, USA) and permeabilized with 0.5% Triton-X (Gibco, USA) for 15 min at room temperature. The cells were blocked with 5% bovine serum albumin (Molecular probes, USA) for 1 h at room temperature, primarily incubated with the rabbit polyclonal anti-ovalbumin antibody (1:250 [1 mg/mL] dilution, Abcam, Cambridge, UK) overnight at 4°C and then secondarily incubated with mouse anti-rabbit fluorescein isothiocyanate (FITC)-conjugated antibody (1:500 [2 mg/mL] dilution, Abcam, UK) for 1 h, and washed with PBS. Immunofluorescence were analyzed under a fluorescence microscope (DMI 6000 B; Leica, Wetzlar, Germany).

Protein from the oviduct magnum tissue (positive control), chicken leg muscle tissue (negative control), DF-1 (negative control), and cOECs at passage 2 were extracted using RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Scientific, USA). The protein concentration was determined with the Bradford assay (Bio-Rad, Hercules, CA, USA). Protein extracts (15 μg) was electrophoresed with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis 4% to 12% gel system (Invitrogen, USA) and transferred to polyvinylidene fluoride (Invitrogen, USA) membrane. The membrane was blocked with 5% skim milk in PBS (Thermo Scientific, USA) for 1 h and then primarily incubated with rabbit polyclonal anti-ovalbumin antibody (1:1,000 [1 mg/mL] dilution, Abcam, UK) and rabbit monoclonal anti-vinculin antibody (1:1,000 [0.054 mg/mL] dilution, Abcam, UK) overnight 4°C. The membrane was washed and then secondarily incubated with mouse anti-rabbit HRP conjugated antibody (1:2,000 [0.4 mg/mL] dilution, Santa Cruz, Dallas, TX, USA) for 30 min at room temperature. Amersham ECL prime (GE Healthcare, Buckinghamshire, UK) substrate was used to visualize the target bands, and the bands were analyzed using EZ-Capture II (Atto, Tokyo, Japan).

cOECs at passage 2 and DF-1 were washed with PBS (Gibco, USA) and recovered through trypsin treatment. The recovered cells were washed with PBS (Gibco, USA), fixed with 2% paraformaldehyde (molecular probes) for 20 min, and permeabilized with 0.02% Triton-X (Gibco, USA) for 15 min. The cells were primarily incubated with the rabbit polyclonal anti-ovalbumin antibody (1:1,000 [1 mg/mL] dilution, Abcam, UK) for 30 min at room temperature and then secondarily incubated with the mouse anti-rabbit FITC antibody (1:1,000 [2 mg/mL] dilution, Abcam, UK) for 20 min at room temperature, only the secondary antibody staining was used as an isotype control. The two abovementioned antibodies were diluted with permeabilization buffer (Gibco, USA). The cells were subsequently washed three times and each 1×10⁴ cells were analyzed using a FACSCalibur (BD Bioscience, Franklin Lakes, NJ, USA).

To validate the ovalbumin promoter activity into cOECs, the mutated 4.4-kb ovalbumin gene promoter (Mut-4.4-kb-pOV) with a 1-kb deletion between the EREs and the 2.8-kb ovalbumin promoter was constructed [10]. This DNA fragment was entirely synthesized, and then cloned into pGL4.11 (luc2p) reporter vector (Promega, Madison, WA, USA). cOECs at passage 4 and DF-1 were transfected with Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, cOECs and DF-1 were seeded into a 12-well cell culture plate, and the transfection was performed at 70% cell density, based on molecular ratio of each vector, namely, mock/pGL4.11 and Mut-4.4-kb-pOV/pGL4.11. For transfection, 1.5 μL of Lipofectamine 3000 reagent suspended in 50 μL of Opti-MEM (Gibco, USA) and 1 μg of each pGL4.11 firefly DNA, 0.01 μg of pGL4.74 (hRluc) renilla DNA (Promega, USA), and 2 μL of p3000 reagent suspended in 50 μL of Opti-MEM were mixed for 20 min at room temperature. The mixture of 100 μL per well was added to each well of the 12-well cell culture plate.

For the reporter analysis, the cell culture medium was removed, and 250 μL of passive lysis buffer (Promega, USA), was added to each well of the 12-well cell culture plate. The plate was then stirred for 20 min for cell lysis, and the lysed cells were transferred to a 1.5 mL Eppendorf tube and centrifuged at 12,000 rpm for 10 min at room temperature. Each lysate of 20 μL was transferred to a 96-well white micro plate (Nunc, Rochester, NY, USA), and the luciferase activities were measured using a Centro LB 960 luminometer (Berthold, Bad Wildbad, Germany). The luciferase ratio for each vector was calculated as follows: (firefly luminescence)/(Renilla luminescence), and the relative luciferase ratio was calculated as follows: (mock/pGL4.11 luciferase ratio)/(Mut-4.4-kb-pOV/pGL4.11 luciferase ratio).

The statistical analyses were conducted using GraphPad Prism statistical software (GraphPad Prism 5.03. software). Student’s t-test was used for the comparison of the relative luciferase ratio of Mut-4.4-kb-pOV/pGL4.11 and mock/pGL4.11, and p-values less than 0.05 was considered to indicate statistical significance. Error bars denote the standard error of the mean.

To obtain cOECs, we isolated oviduct tissue from egg-laying WL hens and analyzed the structure of the inner surface. The length of oviduct tissue separated from the infundibulum to the proximal magnum was approximately 36 cm (Figure 1A), and the inner surface of the magnum epithelium of the chicken oviduct tissue showed thick, anastomosing folds of the mucosa (Figure 1B). The magnum portion of a chicken oviduct tissue was subsequently separated and analyzed by SEM. As shown in Figure 1C, the magnum showed ciliated surface, and ciliated cells were observed in the culture plate after isolating the cells from oviduct tissue (Figure 1D). Through our isolation method, almost of the primary cOECs at passage 0 composed of colony-initiating cells (Figure 2A). The population of cOECs at passage 1 was heterogeneously composed of epithelial-like ciliated nonsecretory cells with a cobble stone shape and fibroblast-like cells (Figure 2B). From at passage 2, the population of epithelial-like ciliated nonsecretory cells continuously diminished, whereas the fibroblast-like cells were maintained at least fifth passages, and most of these cells showed secretory granules (Figure 2C). Furthermore, chromosomal karyotyping of the cultured cOECs showed normal chicken chromosome (2n = 78) (Figure 2D). Altogether, these results demonstrate the efficient isolation and short-term in vitro cultivation of the oviduct epithelial cells.

To characterize the cultured cOECs, we selected a set of chicken oviduct-specific markers. The cDNA from the oviduct magnum tissue, DF-1, and the cOECs were amplified by RT-PCR using chicken oviductal and epithelial cell markers (Table 1). The analysis of chicken oviduct-specific markers revealed that ovalbumin mRNA expression was clearly detected in magnum tissue and the cOECs. However, ESR1 mRNA, but not ovomucoid mRNA expression, was detected in all samples. The analysis with epithelial cell markers showed that occludin and E-cadherin mRNA expression were detected in the oviduct magnum tissue and cOECs, whereas cytokeratin 14 mRNA expression was detected in both DF-1 and cOECs but not in the oviduct magnum tissue. The RT-PCR results are summarized in Table 2. Based on the RT-PCR results, cOECs were qualitatively and quantitatively characterized through immunofluorescence, western blot, and flow cytometry analysis. As shown in Figure 3, the ovalbumin protein was clearly detected in the cOECs but not in DF-1 by immunofluorescence (Figure 3A). This ovalbumin protein was also clearly detected in the cOECs and the oviduct magnum tissue but not in leg muscle tissue and DF-1 by western blot analysis (Figure 3B). In flow cytometry analysis, ovalbumin protein expression in stained DF-1 was hardly detected (6.28%), in unstained DF-1 (0.10%), and in isotype control DF-1 (0.37%). In addition, only 0.11% and 0.18% were detected in unstained and in isotype control cOECs, whereas 79.40% of the stained cOECs showed positive detection of ovalbumin protein (Figure 3C). Taken together, the characterization results clearly demonstrate that ovalbumin protein, chicken oviduct-specific marker, was expressed in in vitro cultured cOECs.

To validate the activity of the ovalbumin promoter in the cOECs, we constructed a reporter vector cloned with a 4.4-kb ovalbumin promoter with an approximately 1-kb deletion between the EREs and the 2.8-kb ovalbumin promoter, and this vector was denoted Mut-4.4-kb-pOV/pGL4.11 (Figure 4A). The Mut-4.4-kb-pOV/pGL4.11 and mock/pGL4.11 vectors were transfected into the cOECs and DF-1, respectively, and the ovalbumin promoter activity in each cell was analyzed through a luciferase assay. The comparative luciferase analysis results showed that Mut-4.4-kb-pOV/pGL4.11 resulted in a 7.1-fold (p<0.001) higher relative luciferase ratio in the cOECs than the mock/pGL4.11 vector. However, the variation in the relative luciferase ratio between the Mut-4.4-kb-pOV/pGL4.11 and mock/pGL4.11 vectors was decreased by 2.0-fold (p<0.001) in DF-1 (Figure 4B). We thus verified that the constructed ovalbumin promoter exhibited specific promoter activity in the cultured cOECs. Collectively, these results demonstrate the successful preparation of cOECs from the area between the infundibulum and magnum in chicken oviduct tissue, the characterization of the cOECs using ovalbumin as a marker of the chicken oviduct, and the analysis of the expression and regulation of specific genes by the ovalbumin promoter in these cells.