Background Although chicken oviduct is a useful model and target tissue

Background Although chicken oviduct is a useful model and target tissue for reproductive biology and transgenesis, little is known because of the highly specific hormonal regulation and the lack of fundamental researches, including lectin-binding activities and glycobiology. on luminal surface in juvenile magnum, but not tubular gland cells. In adult magnum, two types of epithelium and three types of tubular gland cells were observed. qRT-PCR analysis showed that egg-white genes were highly expressed in adult oviduct compared with the juvenile. However, mRNA expressions of culture of chicken oviductal cells. Methods Experimental animals and animal care The care and experimental use of chickens was approved by the PIK-293 Institute of Laboratory Animal Resources, Seoul National University (SNU-070823-5). Chickens were maintained according to a standard management program at the University Animal Farm, Seoul National University, Korea. The procedures for animal management, reproduction, and embryo manipulation adhered to the standard operating protocols of our laboratory. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) The magnum segment of chicken oviducts from juvenile (10-week-old) and actively egg-laying (30-week-old) hens were obtained, fixed primarily at 4C for 2-4 h with modified Karnovsky’s fixative (2% glutaraldehyde and 2% formaldehyde in 0.05 M sodium cacodylate buffer, pH 7.2), washed three times with cacodylate buffer, fixed secondarily for 2 h with 1% osmium tetroxide in cacodylate buffer, and stained overnight with 0.5% uranyl acetate at 4C. To observe specimens for scanning electron microscopy (SEM), samples were dried twice with 100% isoamyl acetate for PIK-293 15 min in a critical point Rabbit Polyclonal to GIPR. dryer, mounted on metal stubs, coated with gold, and observed under field emission (FE)-SEM (SUPRA 55VP; Carl Zeiss). To prepare specimens for transmission electron microscopy (TEM), samples were dehydrated through a graded ethanol series, embedded in Spurr’s resin, and cut on an ultramicrotome (MT-X; RMC, Tucson, AZ, USA). Samples were then stained with 2% uranyl acetate and Reynold’s lead citrate for 7 min each and observed under TEM (LIBRA 120; Carl Zeiss). Total RNA extraction and real-time PCR analysis Total RNA was extracted from the oviduct and muscle samples from juvenile (10-week-old) and egg-laying adult (30-week-old) chickens using TRIzol according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Extracted RNA was quantified using a spectrophotometer and 1 ug of each RNA sample was reverse-transcribed into 20 l of single-stranded cDNA using the Superscript III First-Strand Synthesis System (Invitrogen). Primer sets were synthesized to amplify specific fragments of chicken oviductal transcripts as described in Table ?Table1.1. To analyze the expression patterns PIK-293 of oviduct-specific genes, the iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and EvaGreen (Biotium, Hayward, CA, USA) were used for quantitative RT-PCR. Non-template wells without cDNA were included as negative controls and each test sample was run in triplicate. The PCR amplification was performed at 94C for PIK-293 3 min, followed by 35 cycles at 94C for 30 s, 60C for 30 s, and 72C for 30 s, using a melting curve program (increase PIK-293 in temperature from 55C to 95C at a rate of 0.5C per 10 s) and continuous fluorescence measurement. Relative quantification of gene expression was calculated after normalization of the transcript to GAPDH (endogenous control) and the nonspecific control using the 2-Ct method. The PCR products were also loaded on a 1% agarose gel with ethidium bromide. Table 1 Primer sequences for RT-PCR Immunohistochemistry and lectin staining The oviductal magnum segments of juvenile (10-week-old) and egg-laying adult (30-week-old) chickens were fixed in 4% buffered paraformaldehyde after strong washing with phosphate-buffered saline (PBS). Segments were subsequently embedded into a paraffin block and the paraffin-embedded oviductal tissue was sectioned at a thickness of 6 m. The deparaffinized and rehydrated samples were heated in a microwave for 10 min after immersion in a sodium citrate buffer solution at pH 6.0 for heat-induced epitope retrieval (HIER). For immunohistochemical analysis, samples were permeabilized with 0.1% Triton X-100 in PBS for 5 min and incubated with 0.1% normal goat serum for 1 h to block nonspecific binding..

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