- Email: [email protected]
Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle
Accepted Manuscript Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle M. Elhamouly, N. Isobe, Y. Yoshimura PII:
S0093-691X(17)30290-X
DOI:
10.1016/j.theriogenology.2017.06.015
Reference:
THE 14152
To appear in:
Theriogenology
Received Date: 30 January 2017 Revised Date:
6 June 2017
Accepted Date: 10 June 2017
Please cite this article as: Elhamouly M, Isobe N, Yoshimura Y, Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.06.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
ACCEPTED MANUSCRIPT
Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle
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M. Elhamouly a, N. Isobe a, b and Y. Yoshimura a, b, *
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Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 7398528, Japan
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Research Center for Animal Science, Hiroshima University, Higashi-Hiroshima 7398528, Japan
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Running head: Cyclooxygenases in the hen oviduct
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*Corresponding author:
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Yukinori Yoshimura
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Graduate School of Biosphere Science
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Hiroshima University
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Higashi-Hiroshima 739-8528, Japan
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Prostaglandins (PGs) play important roles in regulation of the functions of the hen oviduct.
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However, little is known about the expression and localization of the rate-limiting
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cyclooxygenases (COX-1 and COX-2) in the oviduct. The aim of this study was to determine
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the COXs expression and localization in the different segments of the oviduct and to
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investigate changes in their expression levels during the ovulatory cycle of laying hens.
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White Leghorn laying hens were killed at 0, 4, 7, 16 and 24 h after oviposition, and samples
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from the infundibulum, magnum, isthmus, uterus, and vagina were collected. Gene and
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protein expressions were examined by real-time PCR and western blot, respectively, for both
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COX-1 and COX-2. Localization of COX-1 and COX-2 in the hen oviduct was determined
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by immunohistochemistry and PCR analysis of samples collected by laser capture
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microdissection (LCM). The expression level of COX-1 was highest in the infundibulum,
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while that of COX-2 was significantly higher in the uterus than in the other segments. The
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expression levels of COX-1 in the infundibulum and COX-2 in the uterus were higher at 0 and
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24 h after oviposition, just prior to subsequent ovulation and oviposition. Western blot
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analysis confirmed the presence of COX-1 and COX-2 in all oviductal segments. The density
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of COX-2 was the highest in the uterus, and did not change during the ovulatory cycle. COX-
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1 and COX-2 were localized in the surface epithelium of all oviductal segments besides the
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uterine tubular glands. We conclude that both COXs are differentially expressed in the
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different oviductal segments with a temporal association to ovulation and oviposition. COX-1
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and COX-2 may play an important role in the infundibulum and uterus, respectively, and
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COX-2 may be one of the factors regulating the induction of oviposition.
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Keywords: cyclooxygenases, prostaglandins, oviduct, laying hen
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1. Introduction Prostaglandins (PGs) are one of the primary factors responsible for regulation of the
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functions of the hen oviduct, such as sperm storage in sperm storage tubules [1], motility of
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the oviduct during transportation of the ovum [2], eggshell calcification [3, 4], and
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oviposition [5-9]. Synthesis of PGs in the oviduct of the laying hen was reported previously
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[4, 10], and maintenance of PGs within physiological levels is important for normal egg
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formation [11]. PGs are supposed to act locally because they interact with their specific
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receptors, while released PGs in the serum are rapidly metabolized [12, 13].
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Cyclooxygenases (COXs; prostaglandin G/H synthase) are primary rate-limiting
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enzymes for the synthesis of PGs and thromboxanes. Two isoenzymes for COXs have been
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identified, namely the constitutive COX-1 and the inducible COX-2, which are encoded by
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the conserved PTGS1 and PTGS2 genes, respectively. COXs are responsible for converting
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arachidonic acid to PGH2, which is a substrate for production of different PGs [14, 15].
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COX-1 and -2 are expressed in the ovary of hens [16], quail [17], and ostrich [18], but little is
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known about their expression or localization in the hen oviduct. It is necessary to characterize
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the COXs expression profiles for better understanding the mechanism by which oviductal
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functions are regulated by PGs. The objective of this study was to characterize the expression
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profiles and localization of COX-1 and COX-2 in the oviduct of laying hens at different times
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during the ovulatory cycle. Specific questions included (1) which tissues expressed the COXs,
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(2) whether COX expression levels differ among the different oviductal segments, and (3)
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whether the expression levels change during an ovulatory cycle in each segment.
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2. Materials and Methods
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2.1. Birds and tissue collection
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ACCEPTED MANUSCRIPT Twenty-five White Leghorn laying hens (46-52 weeks old), regularly laying five or
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more eggs in a sequence, were kept in individual cages under a regimen of 14 h light: 10 h
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dark and provided with commercial feed and water ad libitum. Hens were killed at 0, 4, 7, 16,
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or 24 h after oviposition under anesthesia with sodium pentobarbital (Somnopentyl; Kyoritsu
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Shoji Co., Tokyo, Japan). Samples from different segments of oviduct including the
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infundibulum, magnum, isthmus, uterus, and vagina were collected with confirmation of egg
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location in the oviduct. The egg was absent at 0 h, in the isthmus at 4 h, and in the uterus at 7,
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16, and 24 h after oviposition, whereas the last three times represented the initial, middle and
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final stages of eggshell formation. The handling of birds was performed in accordance with
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guidelines of the Animal Experiment Committee of Hiroshima University.
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2.2. PCR analysis
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2.2.1. RNA extraction
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Tissue samples for extraction of total RNA from the whole mucosa were collected
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from the five different segments of the oviduct at five different time points during the
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ovulatory cycle. Extraction of RNA was performed using Sepasol-RNA I Super (Nacalai
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Tesque Inc., Kyoto, Japan) according to the manufacturer`s instructions. Pelleted RNA was
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dissolved in TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) and kept at -80 °C until
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use.
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tissues (n = 4) at 0 h after oviposition were frozen in isopentane cooled with dry ice. Frozen
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sections (10 µm in thickness) were air-dried and fixed with acetone at −20 °C for 30 s,
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washed with ultrapure water (30 s × two times), and then gradually dehydrated using 70%
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and 95% (v/v) ethanol (30 s each) and 100% ethanol (90 s × two times) on ice. Subsequently,
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they were kept in xylene for 5 min at room temperature and air-dried. The cells of surface
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epithelium and uterine tubular glands were then collected separately, with approximately 200
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spots for one sample, using an LCM system (LM200; Olympus, Tokyo, Japan). Total RNA
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extraction from the collected samples was performed using an RNAGEM Kit (ZyGEM Corp.
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Ltd., Hamilton, New Zealand) according to the manufacturer's instructions.
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2.2.2. Reverse transcription (RT) The RNA samples of the mucosal tissues were treated with RQ1 RNase-free DNase
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(Promega Co., Madison, WI, USA) on a programmable thermal controller (PTC-100; MJ
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Research, Waltham, MA, USA) at 37 °C for 45 min and then at 65 °C for 10 min.
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Concentrations of RNA from mucosal tissue were measured using Gene Quant Pro
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(Amersham Pharmacia Biotech, Cambridge, UK). Then RNA samples were reverse
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transcribed using ReverTra Ace (Toyobo Co. LTD., Osaka, Japan) according to the
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manufacturer’s instructions. The RT reaction mixture (10 µL) consisted of 1 µg of total RNA,
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1× RT buffer, 1 mM deoxynucleotide triphosphate mixture, 20 U of RNase inhibitor, 0.5 µg
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of oligo (dT) 20, and 50 U ReverTra Ace. The RT was performed at 42 °C for 30 min,
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followed by heat inactivation for 5 min at 99 °C using the programmable thermal controller
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(PTC-100; MJ Research).
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The RNA samples collected by LCM were also treated with RQ1 RNase-free DNase
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(Promega Co.) as mentioned above. The RT was performed with same conditions as above
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using the programmable thermal controller (PTC-100; MJ Research).
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2.2.3. Polymerase chain reaction (PCR)
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The PCR was performed using Takara Ex Taq Kit (Takara Bio. Inc., Shiga, Japan)
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on a thermal controller (PTC-100; MJ Research). Primer sequences of COX-1 (XM_425326)
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were
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TGTAGCCGTACTGGGAGTTGAA; COX-2 (NM_001167719) primers were forward =
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CTGCTCCCTCCCATGTCAGA and reverse = CACGTGAAGAATTCCGGTGTT; and
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RPS-17 (NM_204217.1) primers were forward = AAGCTGCAGGAGGAGGAGAGG and
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reverse= GGTTGGACAGGCTGCCGAAGT. The reaction mixture (25 µL) consisted of 0.5
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TCAGGTGGTTCTGGGACATCA
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reverse
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Takara Ex Taq, and 0.4 µM of each primer. PCR products were electrophoretically separated
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on a 2% (w/v) agarose gel with 0.6 µg/mL ethidium bromide and examined on a
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transilluminator (NTM-10E; UVP LLC, Upland, CA, USA).
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2.2.4. Quantitative real-time PCR (qRT-PCR)
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The expression levels of target genes within each oviductal segment at different
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times of an ovulatory cycle were analyzed by real-time PCR using a Roche Light Cycler
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Nano System (Roche Applied Science, Indianapolis, IN, USA). The reaction mixture (10 µL)
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consisted of 0.5 µL cDNA, 1× Thunderbird SYBR qPCR Mix (Toyobo Co. LTD., Osaka,
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Japan) and 250 nM of each primer. Amplification conditions for COX-1 and COX-2 were
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50 °C for 2 min, 95 °C for 2 min, 50 cycles at 95 °C for 10 s, and 60 °C for 40 s.
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Amplification conditions RPS-17 were 45 cycles at 95 °C for 30 s and 62 °C for 30 s.
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Real-time PCR data were analyzed by the 2-∆∆CT method to calculate the relative
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level of expression in each sample using the RPS-17 expression as the internal control. The
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results were shown as the expression level relative to the standard sample (a sample from the
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magnum at 4 h after oviposition). The expression levels were compared among the five
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oviductal segments at each of the five time points of ovulatory cycle, and among the five
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different time points of an ovulatory cycle within each of the five oviductal segments.
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2.3. Immunohistochemistry
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Samples from the middle part of each oviductal segment collected at 24 h after
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oviposition were fixed in 10% (v/v) formalin in phosphate buffered saline (PBS) (n = 4).
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They were processed for paraffin sections (4 µm in thickness). Immunostaining for COX-1
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and COX-2 was performed using monoclonal antibody to human COX-1 (Abcam Inc.,
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Cambridge MA, UK), and polyclonal antibody to human COX-2 (Cayman Chemical, Ann
ACCEPTED MANUSCRIPT Arbor, MI, USA), which have been used to identify COX-1 and COX-2 in the hen ovary by
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Eilati et al. [19] and Hales at al. [16], respectively. Antigen retrieval was performed by
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heating at 98 °C in 10 mM trisodium citrate, pH 6.0, with 0.05% (v/v) Tween-20 for 30 and
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10 min for COX-1 and COX-2, respectively. Endogenous peroxidases were quenched using
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0.3% (v/v) H2O2 in methanol for 15 min. Endogenous biotin was blocked using Avidin/Biotin
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Blocking kit (Vector Laboratories, Burlingame, CA, USA). Sections were incubated with 1%
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(w/v) bovine serum albumin (BSA) (Sigma-Aldrich Japan K.K., Tokyo, Japan) for 1 h,
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followed by monoclonal anti-COX-1 or polyclonal anti-COX-2 antibodies overnight at 4 °C.
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The COX-1 and COX-2 antibodies were diluted in PBS, which contained 1% (w/v) BSA and
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0.1% (v/v) tween-20. The final concentrations of the COX-1 and COX-2 antibody solutions
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were 0.15 and 1 µg/mL, respectively. Normal rabbit IgG (Santa Cruz Biotechnology, Inc.
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Texas, USA) diluted in the same way used for negative control staining. After washing by
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Tris-buffered saline, sections were incubated with biotinylated anti-rabbit IgG (1:200) for 30
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min, followed by incubation with avidin-biotin complex (1:50) (Vector Laboratories) for 30
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min. The immunoreaction products were visualized using a reaction mixture of 0.02% (w/v)
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3, 3´-diaminobenzidine-4HCl and 0.05% (v/v) H2O2, counterstained with hematoxylin, and
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then examined under a light microscope.
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2.4. Western blot
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Mucosal tissues from different segments of the oviduct were homogenized in lysis
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buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 10%
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(v/v) glycerol, 0.1% (w/v) SDS, and 10 mM PMSF) using a POLYTRON® homogenizer
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(Kinematica AG, Tokyo-to, Japan), and then kept on ice for 30 min. They were then
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centrifuged at 12,500 ×g for 15 min at 4 °C, and the supernatant was kept at – 80 °C until use.
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Protein concentration was measured using protein assay reagent (Bio-Rad, Lab., Hercules,
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CA, USA) using BSA for making the standard curve. Samples were mixed with Laemmli
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bromophenol blue and 150 mM Tris-HCl, pH 7.0), and boiled for 10 min. Samples containing
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20 µg of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel
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electrophoresis (SDS-PAGE; 10% separating gel and 4% stacking gel) using 80 V in the
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stacking gel and 120 V in the separating gel.
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After SDS-PAGE, the proteins in the gel were electrophoretically transferred onto a
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0.45 µm Amersham Hybond-C® nitrocellulose membrane at 45 mA for 3 h using a transfer
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buffer (25 mM Tris base, 192 mM glycine and 20% (v/v) methanol, pH 8.3). The membrane
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was incubated with 0.25% (w/v) BSA in washing buffer (20 mM Tris-HCl, pH 7.5, 150 mM
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NaCl, and 0.1% (v/v) tween-20) for 1 h at room temperature to block non-specific binding.
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Membranes were then incubated with monoclonal rabbit anti-human COX-1 (Abcam) or
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monoclonal mouse anti-chicken COX-2 (BD Transduction Lab., Lexington, KY, USA), at
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concentrations of 0.3 µg/mL and 0.25 µg/mL, respectively. Membranes were also incubated
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with anti-chicken β-actin (Santa Cruz Biotechnol., Inc. Texas, USA) diluted at 1:5000, for
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normalization of the density of bands at the final analysis. After washing with the washing
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buffer (10 min x 3 times), the membranes were incubated with peroxidase-labeled secondary
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IgG (GE Healthcare, Buckinghamshire, UK) diluted at 1:2000 in washing buffer for 1 h at
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room temperature. The membranes were washed (10 min × 3 times), and the
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immunoprecipitates on the membrane were treated by Amersham ECLTM Western blotting
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detection reagents (GE Healthcare) for 1 min. The densities of the bands were analyzed using
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an ATTO cooled CCD camera system EZ-Capture II (ATTO Corporation, Tokyo, Japan).
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Optical densities of bands were measured using CS Analyzer® 3.0 software (ATTO, HQ:
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Bunkyoku, Tokyo, Japan).
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2.5. Statistical analysis
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ACCEPTED MANUSCRIPT The significance of differences in gene expression and protein densities among
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different oviductal segments and among different times in an ovulatory cycle was analyzed
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using One-way ANOVA followed by Tukey’s test using IBM SPSS Statistics Desktop 22.0.
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P values of <0.05 were considered significant.
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3. Results
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3.1. COX-1 and COX-2 expression
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Figure 1 shows the expression profiles of COX-1 in different oviductal segments at
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different times after oviposition. The expression level of COX-1 was the highest in the
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infundibulum at all times (Fig. 1A). No significant differences were found among its
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expression levels in the magnum, isthmus, uterus, and vagina at each time point, except at 4 h
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after oviposition when the level was higher in the vagina than in the isthmus (Fig. 1A). As
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shown in Fig. 1B, the COX-1 expression level in the infundibulum was higher at 0 and 24 h
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than at 7 and 16 h after oviposition. In the magnum, COX-1 expression was higher at 24 h
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than at 7 and 16 h after oviposition. In the isthmus, where the expression level was the lowest
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among all oviductal segments, it was higher at 0 h than at 4, 7, and 16 h after oviposition (Fig.
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1B). In the uterus, the expression level was lower at 16 h than the other time points. The
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expression level in the vagina was lower at 16 and 24 h than at 4 h after oviposition (Fig. 1B).
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Figure 2 shows the expression profiles of COX-2 in the different oviductal segments
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at different times after oviposition. The expression level of COX-2 was the highest in the
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uterus relative to the other segments at all times after oviposition except at 4 and 16 h when
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the expression level was not significantly different from that in the vagina (Fig. 2A). Figure
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2B shows the changes in expression level of COX-2 in the different oviductal segments
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during an ovulatory cycle. The expression level in the infundibulum was higher at 4 h than at
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24 h after oviposition, whereas there was no difference in the expression levels among
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different time points in the magnum and isthmus. In the uterus, the COX-2 expression level
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was significantly higher at 24 h than at 4 and 16 h after oviposition. In contrast, the
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expression level in the vagina was lower at 24 h than at 4 h after oviposition.
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3.2. COX-1 and COX-2 protein contents Western blot analysis showed that COX-1 and COX-2 proteins existed in the
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different segments of the oviduct (Fig. 3A). Image analysis showed the density of COX-2
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protein was higher in the uterus than in the other segments (data not shown; analysis was
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performed only on the COX-2 bands without normalization by β-actin density). Figure 3B
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shows the western blot of COX-1 and COX-2 in the uterine mucosa at different times after
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oviposition. There were no significant differences in density among different times after
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oviposition within COX-1 or COX-2 proteins (Fig. 3C, D).
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3.3. Localization of COX-1 and COX-2
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Immunolocalization of COX-1 in the oviduct at 24 h after oviposition is shown in
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Fig. 4A- E. Immunoreaction for COX-1 was localized in the mucosal epithelium of all
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oviductal segments, tubular gland cells of the uterus, vascular endothelium, leukocytes, and
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smooth muscles in the oviduct at different times after oviposition. Nuclear and cytoplasmic
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staining patterns were frequently demonstrated in those cells (Fig. 4A-E).
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COX-2 immunoreaction signals at 24 h after oviposition are shown in Fig. 5A-E.
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They were mainly localized in the surface epithelium of all segments. Smooth muscles,
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vascular endothelium, and leukocytes in all segments showed positive staining for COX-2.
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Weak staining for COX-2 was found in the tubular gland cells of the uterus (Fig. 5D). Those
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immunoreactions for COX-2 were generally identified in the cytoplasm and around nuclei.
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No staining was observed on the sections incubated with normal rabbit IgG in place of COX-1 or COX-2 antibodies (Fig. 5F).
ACCEPTED MANUSCRIPT The expression of both COX-1 and COX-2 was identified in the whole mucosa as
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well as in the uterine surface epithelium and tubular gland cells isolated by the LCM from the
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uterus at 0 h after oviposition (Fig. 6).
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4. Discussion
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We report here the expression profiles and localization of COXs that are responsible
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for PGs synthesis in the hen oviduct. The major findings are that (1) COX-1 expression was
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the highest in the infundibulum, and was higher around the time of oviposition and ovulation
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(0 and 24 h after oviposition) than the other times; (2) expression of COX-2 was the highest
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in the uterus, and the mRNA expression was the highest immediately prior to oviposition (24
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h after the previous oviposition); and (3) The surface epithelium of all segments was
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immunopositive for both COXs. Asboth et al. [10] showed that PGE2 and PGF2α were
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produced in the hen oviduct and that their amounts were higher in the infundibulum, uterus,
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and vagina than in the magnum and isthmus. The current study supports their report, since the
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expression of COX-1 was the highest in the infundibulum, while COX-2 expression and its
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protein contents were the highest in the uterus followed by the vagina.
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Immunoreactive COX-1 and COX-2 were localized in the surface epithelium,
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capillary endothelial cells, and leukocytes in the lamina propria in all oviductal segments.
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The expression of both COXs in the surface epithelium of the uterus was supported by RT-
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PCR using RNA specifically isolated from those tissues by LCM. In the tubular gland cells of
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the uterus, obvious immunoreaction to COX-1 was identified, but COX-2 showed weak
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immunoreaction. However, the expression of both COX-1 and COX-2 was clearly identified
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in the tubular gland cells isolated by LCM. Thus, the uterine tubular gland cells may have the
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ability to synthesize both COX-1 and COX-2, although the amount of COX-2 protein may be
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small in those cells. Reports on COXs localization in the avian oviduct are not available, but
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studies on the bovine oviduct suggested that COXs regulated its functions by the produced
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ACCEPTED MANUSCRIPT PGs [20]. In the current study, nuclear, perinuclear, and cytoplasmic staining patterns for
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COXs were shown. Similar staining patterns have been shown in the ovary of chicken [16]
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and of other avian species such as quail [17] and ostrich [18]. Subcellular localization of
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COXs is frequently reported to be on the luminal surface of the endoplasmic reticulum and
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the nuclear envelope [21].
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In the infundibulum, COX-1 was identified by western blot and its mRNA
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expression level was higher than in the other segments. Although the changes in the COX-1
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protein density during an ovulatory cycle remain unknown, the expression level was higher at
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0 and 24 h after oviposition. Although reports showing such results in birds are not available,
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there is a report showing that COX-1 mRNA expression in the bovine oviduct was highest
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around ovulation [20]. Thus, we suggest that COX-1 may play a vital role in ovulation by
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regulating the infundibulum, showed an enhanced contractility and close to the ovary around
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ovulation so as to cause ovulation and capture the released ovum [22].
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PGs are important for oviposition in the hens [8, 23], as blocking of their synthesis
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by treatment with indomethacin delayed oviposition [24]. The serum levels of PGs increased
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around the time of oviposition [5] but their source is still controversial. The concentration of
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PGF2α in the uterus showed a peak around the time of oviposition, suggesting a local
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synthesis of PGs [7]. The ruptured ovarian follicles were also reported to play roles for
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oviposition in laying hens [25] by the production of PGs through COX-1 [16]. Contrary to the
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chickens, it was suggested that PGs have no significant roles in neither ovulation nor
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oviposition in the ostrich [18]. The current study shows that COX-1 and COX-2 are
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expressed in the different segments of the oviduct including the uterus, besides that COX-2
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gene expression and its protein density were higher in the uterus than in the other oviductal
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segments. These results suggest that COX-2 may have a role in the regulation of the functions
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of the uterus. It has been reported that COX-2 is inducible by many factors such as
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ACCEPTED MANUSCRIPT inflammatory cytokines [14], mechanical stress [26] or stretching [27]. Thus, such factors
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may control COX-2 expression in the uterus of the laying hen, and affect the local synthesis
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of PGs and oviposition as a consequence, but this may require an additional study to be
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evaluated. Although COX-2 expression in the uterus was up-regulated at 24 h after
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oviposition, the COX-2 protein density did not change during the ovulatory cycle, and the
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reason for this is unknown. Kang et al. [28] mentioned that COX-2 mRNA and protein have
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short half-lives compared with those of COX-1. We assume that COX-2 protein turnover, i.e.,
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replacement of the COX-2 protein with fresh protein, may occur rapidly, and thus, the COX-
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2 protein density did not significantly change at 24 h after oviposition. The fresh COX-2 may
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be responsible for the increase in PGS synthesis that then influences oviposition in the uterus.
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Monocytes are known to express COXs [29], and our previous study showed that the
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monocyte population in the vagina was higher than in the other oviductal segments [30]. The
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expression level of COX-2 was relatively higher in the vagina until 4 h after oviposition. This
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may be associated with the higher population of leukocytes expressing COX-2 in the vagina
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(Fig. 5E).
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In conclusion, we suggest that the oviductal mucosa synthesizes COXs, providing
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the potential ability to synthesize PGs. COX-1 may have an important role in mediating PGs
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production required for the activity of the infundibulum in engulfing the ovulated ovum.
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COX-2 may have an important role in the production of PGs necessary for performing the
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functions of the uterus including oviposition.
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Acknowledgements
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This work was supported by a Grant-in-Aid for Scientific Research from JSPS (No. 25292161) to Y.Y.
ACCEPTED MANUSCRIPT References
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[1] Sasanami T, Izumi S, Sakurai N, Hirata T, Mizushima S, Matsuzaki M, et al. A unique
318
mechanism of successful fertilization in a domestic bird. Sci Rep. 2015;5:7700.
319
[2] Wechsung E, Houvenaghel A. The influence of prostaglandins on the in vitro motility of
320
the oviduct of the laying hen. Zentralbl Veterinarmed A. 1975;22:684-8.
321
[3] Lundholm CD. DDE-induced eggshell thinning in birds: effects of p,p'-DDE on the
322
calcium and prostaglandin metabolism of the eggshell gland. Comp Biochem Physiol C
323
Pharmacol Toxicol Endocrinol. 1997;118:113-28.
324
[4] Lundholm CE, Bartonek M. Inhibition of eggshell formation in domestic fowl by
325
indomethacin: relation to calcium and prostaglandin metabolism in the eggshell gland
326
mucosa. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1992;102:379-83.
327
[5] Olson DM, Shimada K, Etches RJ. Prostaglandin concentrations in peripheral plasma and
328
ovarian and uterine plasma and tissue in relation to oviposition in hens. Biol Reprod.
329
1986;35:1140-6.
330
[6] Takahashi T, Tajima H, Nakagawa-Mizuyachi K, Nakayama H, Kawashima M. Changes
331
in prostaglandin F2 alpha receptor bindings in the hen oviduct uterus before and after
332
oviposition. Poult Sci. 2011;90:1767-73.
333
[7] Takahashi T, Ogawa H, Inaba R, Kawashima M. Changes in prostaglandin F
334
concentration in the uterus (shell gland) of the hen oviduct in relation to oviposition and
335
estrogen. Poult Sci. 2004;83:1745-9.
336
[8] Hargrove TL, Ottinger MA. Induced oviposition of precalcified eggs following
337
prostaglandin administration. Poult Sci. 1992;71:548-52.
338
[9] Hertelendy F, Biellier HV. Evidence for a physiological role of prostaglandins in
339
oviposition by the hen. J Reprod Fertil. 1978;53:71-4.
AC C
EP
TE D
M AN U
SC
RI PT
316
ACCEPTED MANUSCRIPT [10] Asboth G, Todd H, Toth M, Hertelendy F. PGE2 binding, synthesis, and distribution in
341
hen oviduct. Am J Physiol. 1985;248:E80-8.
342
[11] Hester PY, Newlon NF, Klingensmith PM. Plasma, follicular, and uterine levels of
343
prostaglandins in chickens laying soft-shelled and shell-less eggs. Poult Sci. 1991;70:1585-93.
344
[12] Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and
345
functions. Physiol Rev. 1999;79:1193-226.
346
[13] Coleman RA, Smith WL, Narumiya S. International Union of Pharmacology
347
classification of prostanoid receptors: properties, distribution, and structure of the receptors
348
and their subtypes. Pharmacol Rev. 1994;46:205-29.
349
[14] Rouzer CA, Marnett LJ. Cyclooxygenases: structural and functional insights. J Lipid Res.
350
2009;50 Suppl:S29-34.
351
[15] Chandrasekharan NV, Simmons DL. The cyclooxygenases. Genome Biol. 2004;5:241-
352
41.
353
[16] Hales DB, Zhuge Y, Lagman JA, Ansenberger K, Mahon C, Barua A, et al.
354
Cyclooxygenases expression and distribution in the normal ovary and their role in ovarian
355
cancer in the domestic hen (Gallus domesticus). Endocrine. 2008;33:235-44.
356
[17] Rodler D, Sinowatz F. Expression of prostaglandin-synthesizing enzymes
357
(cyclooxygenase 1, cyclooxygenase 2) in the ovary of the quail (Coturnix japonica). Folia
358
Biol (Praha). 2015;61:125-33.
359
[18] Rodler D, Sinowatz F. Expression of prostaglandin synthesizing enzymes
360
(cyclooxygenase 1 and cyclooxygenase 2) in the ovary of the ostrich (Struthio camelus). Acta
361
Histochemica. 2015;117:69-75.
362
[19] Eilati E, Hales K, Zhuge Y, Ansenberger Fricano K, Yu R, van Breemen RB, et al.
363
Flaxseed enriched diet-mediated reduction in ovarian cancer severity is correlated to the
364
reduction of prostaglandin E(2) in laying hen ovaries. Prostaglandins Leukot Essent Fatty
365
Acids. 2013;89:179-87.
AC C
EP
TE D
M AN U
SC
RI PT
340
ACCEPTED MANUSCRIPT [20] Odau S, Gabler C, Holder C, Einspanier R. Differential expression of cyclooxygenase 1
367
and cyclooxygenase 2 in the bovine oviduct. J Endocrinol. 2006;191:263-74.
368
[21] Spencer AG, Woods JW, Arakawa T, Singer, II, Smith WL. Subcellular localization of
369
prostaglandin endoperoxide H synthases-1 and -2 by immunoelectron microscopy. J Biol
370
Chem. 1998;273:9886-93.
371
[22] Warren DC, Scott HM. Ovulation in the domestic hen. Science. 1934;80:461-62.
372
[23] Hertelendy F, Yeh M, Biellier HV. Induction of oviposition in the domestic hen by
373
prostaglandins. Gen Comp Endocrinol. 1974;22:529-31.
374
[24] Rzasa J, Paczoska-Eliasiewicz H. Effect of indomethacin and vasotocin on oviposition in
375
the hen (Gallus domesticus). Acta Physiol Pol. 1989;40:111-5.
376
[25] Rothchild I, Fraps RM. On the function of the ruptured ovarian follicle of the domestic
377
fowl. Proc Soc Exp Biol Med. 1944;56:79-82.
378
[26] Fermor B, Weinberg JB, Pisetsky DS, Misukonis MA, Fink C, Guilak F. Induction of
379
cyclooxygenase-2 by mechanical stress through a nitric oxide-regulated pathway.
380
Osteoarthritis Cartilage. 2002;10:792-8.
381
[27] Norregaard R, Jensen BL, Topcu SO, Nielsen SS, Walter S, Djurhuus JC, et al.
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Cyclooxygenase type 2 is increased in obstructed rat and human ureter and contributes to
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pelvic pressure increase after obstruction. Kidney Int. 2006;70:872-81.
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[28] Kang Y-J, Mbonye UR, DeLong CJ, Wada M, Smith WL. Regulation of intracellular
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cyclooxygenase levels by gene transcription and protein degradation. Prog Lipid Res.
386
2007;46:108-25.
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[29] Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of
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prostaglandin synthesis and inhibition. Pharmacol Rev. 2004;56:387-437.
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[30] Zheng WM, Yoshimura Y. Localization of macrophages in the chicken oviduct: effects
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of age and gonadal steroids. Poult Sci. 1999;78:1014-8.
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Fig. 1. Expression of COX-1 in different segments of oviduct at different times after
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oviposition. (A) Comparison of COX-1 expression among different segments at each time
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after oviposition, (B) Changes in COX-1 expression at different times (0 to 24 h) after
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oviposition within each segment of the oviduct. Values are mean ± SE of the ratio of COX-1
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to RPS-17 expression (n = 5). Bars with different letters are significantly different (P < 0.05).
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Fig. 2. Expression of COX-2 in different segments of the oviduct at different times after
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oviposition. (A) Comparison of COX-2 expression among different segments at each time
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after oviposition, (B) Changes in COX-2 expression at different times (0 to 24 h) after
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oviposition within each segment of the oviduct. Values are mean ± SE of the ratio of COX-2
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to RPS-17 expression (n = 5). Bars with different letters are significantly different (P < 0.05).
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Fig. 3. Western blot of COX-1 and COX-2 in the oviduct of laying hens. (A) Western blot of
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COX-1 and COX-2 in the mucosa of different oviductal segments at 4 h after oviposition. (B)
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Western blot of COX-1 and COX-2 in the uterine mucosa at different times after oviposition.
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(C and D) Densities of immunoreaction bands of COX-1 and COX-2 in the uterine mucosa
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collected at different times (0 to 24 h) after oviposition, respectively. Values are mean ± SE
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(n = 4) of the ratios of COXs to β-actin (P < 0.05).
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Fig. 4. Immunostaining for COX-1 in the different segments of the oviduct. (A)
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Infundibulum, (B) Magnum, (C) Isthmus, (D) Uterus and (E) Vagina at 24 h after oviposition.
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Immunoreaction products are observed in the cytoplasm and nuclei of surface epithelium (e),
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uterine tubular glands (g), smooth muscles (m), leukocytes (arrow) and vascular endothelium
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(arrowhead). Scale bar= 100 µm.
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Fig. 5. Immunostaining for COX-2 in the different segments of the oviduct. (A)
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Infundibulum, (B) Magnum, (C) Isthmus, (D) Uterus and (E) Vagina at 24 h after oviposition.
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ACCEPTED MANUSCRIPT Immunoreaction products are observed in the surface epithelium (e), uterine tubular glands
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(g), smooth muscles (m), leukocytes (arrow), and blood vessels (arrowhead). (F) Negative
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control section incubated with normal rabbit IgG in place of the primary antibody. Scale bar=
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100 µm.
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Fig. 6. RT-PCR products of COX-1 and COX-2, in the uterine surface epithelium (E), tubular
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glands (G) and whole mucosa (M) at 0 h after oviposition. The RNA samples of the surface
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epithelium and tubular glands were collected using LCM.
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A 10
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We examined the expression and localization of the two isoforms of cyclooxygenase in the different segments of the oviduct of laying hens at different time points in the ovulatory cycle.
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COX-1 and COX-2 are localized in the surface epithelium of all oviductal segments besides the uterine tubular glands.
COX-1 gene expression is the highest at the infundibulum than in the other oviductal
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segments.
COX-2 is expressed significantly higher in the uterus than in the other oviductal
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COX-1 and COX-2 gene expression may influence the activity of the infundibulum
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during ovulation and the functions of the uterus till oviposition, respectively.
S0093-691X(17)30290-X
DOI:
10.1016/j.theriogenology.2017.06.015
Reference:
THE 14152
To appear in:
Theriogenology
Received Date: 30 January 2017 Revised Date:
6 June 2017
Accepted Date: 10 June 2017
Please cite this article as: Elhamouly M, Isobe N, Yoshimura Y, Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.06.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Revised
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Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle
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M. Elhamouly a, N. Isobe a, b and Y. Yoshimura a, b, *
3 a
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 7398528, Japan
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Research Center for Animal Science, Hiroshima University, Higashi-Hiroshima 7398528, Japan
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Running head: Cyclooxygenases in the hen oviduct
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*Corresponding author:
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Yukinori Yoshimura
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Graduate School of Biosphere Science
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Hiroshima University
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Higashi-Hiroshima 739-8528, Japan
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Prostaglandins (PGs) play important roles in regulation of the functions of the hen oviduct.
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However, little is known about the expression and localization of the rate-limiting
26
cyclooxygenases (COX-1 and COX-2) in the oviduct. The aim of this study was to determine
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the COXs expression and localization in the different segments of the oviduct and to
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investigate changes in their expression levels during the ovulatory cycle of laying hens.
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White Leghorn laying hens were killed at 0, 4, 7, 16 and 24 h after oviposition, and samples
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from the infundibulum, magnum, isthmus, uterus, and vagina were collected. Gene and
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protein expressions were examined by real-time PCR and western blot, respectively, for both
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COX-1 and COX-2. Localization of COX-1 and COX-2 in the hen oviduct was determined
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by immunohistochemistry and PCR analysis of samples collected by laser capture
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microdissection (LCM). The expression level of COX-1 was highest in the infundibulum,
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while that of COX-2 was significantly higher in the uterus than in the other segments. The
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expression levels of COX-1 in the infundibulum and COX-2 in the uterus were higher at 0 and
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24 h after oviposition, just prior to subsequent ovulation and oviposition. Western blot
38
analysis confirmed the presence of COX-1 and COX-2 in all oviductal segments. The density
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of COX-2 was the highest in the uterus, and did not change during the ovulatory cycle. COX-
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1 and COX-2 were localized in the surface epithelium of all oviductal segments besides the
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uterine tubular glands. We conclude that both COXs are differentially expressed in the
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different oviductal segments with a temporal association to ovulation and oviposition. COX-1
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and COX-2 may play an important role in the infundibulum and uterus, respectively, and
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COX-2 may be one of the factors regulating the induction of oviposition.
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Keywords: cyclooxygenases, prostaglandins, oviduct, laying hen
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1. Introduction Prostaglandins (PGs) are one of the primary factors responsible for regulation of the
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functions of the hen oviduct, such as sperm storage in sperm storage tubules [1], motility of
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the oviduct during transportation of the ovum [2], eggshell calcification [3, 4], and
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oviposition [5-9]. Synthesis of PGs in the oviduct of the laying hen was reported previously
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[4, 10], and maintenance of PGs within physiological levels is important for normal egg
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formation [11]. PGs are supposed to act locally because they interact with their specific
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receptors, while released PGs in the serum are rapidly metabolized [12, 13].
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Cyclooxygenases (COXs; prostaglandin G/H synthase) are primary rate-limiting
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enzymes for the synthesis of PGs and thromboxanes. Two isoenzymes for COXs have been
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identified, namely the constitutive COX-1 and the inducible COX-2, which are encoded by
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the conserved PTGS1 and PTGS2 genes, respectively. COXs are responsible for converting
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arachidonic acid to PGH2, which is a substrate for production of different PGs [14, 15].
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COX-1 and -2 are expressed in the ovary of hens [16], quail [17], and ostrich [18], but little is
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known about their expression or localization in the hen oviduct. It is necessary to characterize
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the COXs expression profiles for better understanding the mechanism by which oviductal
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functions are regulated by PGs. The objective of this study was to characterize the expression
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profiles and localization of COX-1 and COX-2 in the oviduct of laying hens at different times
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during the ovulatory cycle. Specific questions included (1) which tissues expressed the COXs,
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(2) whether COX expression levels differ among the different oviductal segments, and (3)
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whether the expression levels change during an ovulatory cycle in each segment.
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2. Materials and Methods
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2.1. Birds and tissue collection
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ACCEPTED MANUSCRIPT Twenty-five White Leghorn laying hens (46-52 weeks old), regularly laying five or
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more eggs in a sequence, were kept in individual cages under a regimen of 14 h light: 10 h
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dark and provided with commercial feed and water ad libitum. Hens were killed at 0, 4, 7, 16,
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or 24 h after oviposition under anesthesia with sodium pentobarbital (Somnopentyl; Kyoritsu
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Shoji Co., Tokyo, Japan). Samples from different segments of oviduct including the
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infundibulum, magnum, isthmus, uterus, and vagina were collected with confirmation of egg
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location in the oviduct. The egg was absent at 0 h, in the isthmus at 4 h, and in the uterus at 7,
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16, and 24 h after oviposition, whereas the last three times represented the initial, middle and
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final stages of eggshell formation. The handling of birds was performed in accordance with
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guidelines of the Animal Experiment Committee of Hiroshima University.
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2.2. PCR analysis
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2.2.1. RNA extraction
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Tissue samples for extraction of total RNA from the whole mucosa were collected
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from the five different segments of the oviduct at five different time points during the
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ovulatory cycle. Extraction of RNA was performed using Sepasol-RNA I Super (Nacalai
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Tesque Inc., Kyoto, Japan) according to the manufacturer`s instructions. Pelleted RNA was
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dissolved in TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) and kept at -80 °C until
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tissues (n = 4) at 0 h after oviposition were frozen in isopentane cooled with dry ice. Frozen
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sections (10 µm in thickness) were air-dried and fixed with acetone at −20 °C for 30 s,
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washed with ultrapure water (30 s × two times), and then gradually dehydrated using 70%
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and 95% (v/v) ethanol (30 s each) and 100% ethanol (90 s × two times) on ice. Subsequently,
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they were kept in xylene for 5 min at room temperature and air-dried. The cells of surface
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epithelium and uterine tubular glands were then collected separately, with approximately 200
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spots for one sample, using an LCM system (LM200; Olympus, Tokyo, Japan). Total RNA
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extraction from the collected samples was performed using an RNAGEM Kit (ZyGEM Corp.
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Ltd., Hamilton, New Zealand) according to the manufacturer's instructions.
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2.2.2. Reverse transcription (RT) The RNA samples of the mucosal tissues were treated with RQ1 RNase-free DNase
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(Promega Co., Madison, WI, USA) on a programmable thermal controller (PTC-100; MJ
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Research, Waltham, MA, USA) at 37 °C for 45 min and then at 65 °C for 10 min.
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Concentrations of RNA from mucosal tissue were measured using Gene Quant Pro
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(Amersham Pharmacia Biotech, Cambridge, UK). Then RNA samples were reverse
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transcribed using ReverTra Ace (Toyobo Co. LTD., Osaka, Japan) according to the
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manufacturer’s instructions. The RT reaction mixture (10 µL) consisted of 1 µg of total RNA,
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1× RT buffer, 1 mM deoxynucleotide triphosphate mixture, 20 U of RNase inhibitor, 0.5 µg
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of oligo (dT) 20, and 50 U ReverTra Ace. The RT was performed at 42 °C for 30 min,
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followed by heat inactivation for 5 min at 99 °C using the programmable thermal controller
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(PTC-100; MJ Research).
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The RNA samples collected by LCM were also treated with RQ1 RNase-free DNase
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(Promega Co.) as mentioned above. The RT was performed with same conditions as above
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using the programmable thermal controller (PTC-100; MJ Research).
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2.2.3. Polymerase chain reaction (PCR)
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The PCR was performed using Takara Ex Taq Kit (Takara Bio. Inc., Shiga, Japan)
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on a thermal controller (PTC-100; MJ Research). Primer sequences of COX-1 (XM_425326)
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were
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TGTAGCCGTACTGGGAGTTGAA; COX-2 (NM_001167719) primers were forward =
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CTGCTCCCTCCCATGTCAGA and reverse = CACGTGAAGAATTCCGGTGTT; and
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RPS-17 (NM_204217.1) primers were forward = AAGCTGCAGGAGGAGGAGAGG and
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reverse= GGTTGGACAGGCTGCCGAAGT. The reaction mixture (25 µL) consisted of 0.5
forward
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TCAGGTGGTTCTGGGACATCA
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reverse
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Takara Ex Taq, and 0.4 µM of each primer. PCR products were electrophoretically separated
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on a 2% (w/v) agarose gel with 0.6 µg/mL ethidium bromide and examined on a
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transilluminator (NTM-10E; UVP LLC, Upland, CA, USA).
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2.2.4. Quantitative real-time PCR (qRT-PCR)
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The expression levels of target genes within each oviductal segment at different
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times of an ovulatory cycle were analyzed by real-time PCR using a Roche Light Cycler
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Nano System (Roche Applied Science, Indianapolis, IN, USA). The reaction mixture (10 µL)
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consisted of 0.5 µL cDNA, 1× Thunderbird SYBR qPCR Mix (Toyobo Co. LTD., Osaka,
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Japan) and 250 nM of each primer. Amplification conditions for COX-1 and COX-2 were
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50 °C for 2 min, 95 °C for 2 min, 50 cycles at 95 °C for 10 s, and 60 °C for 40 s.
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Amplification conditions RPS-17 were 45 cycles at 95 °C for 30 s and 62 °C for 30 s.
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Real-time PCR data were analyzed by the 2-∆∆CT method to calculate the relative
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level of expression in each sample using the RPS-17 expression as the internal control. The
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results were shown as the expression level relative to the standard sample (a sample from the
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magnum at 4 h after oviposition). The expression levels were compared among the five
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oviductal segments at each of the five time points of ovulatory cycle, and among the five
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different time points of an ovulatory cycle within each of the five oviductal segments.
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2.3. Immunohistochemistry
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Samples from the middle part of each oviductal segment collected at 24 h after
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oviposition were fixed in 10% (v/v) formalin in phosphate buffered saline (PBS) (n = 4).
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They were processed for paraffin sections (4 µm in thickness). Immunostaining for COX-1
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and COX-2 was performed using monoclonal antibody to human COX-1 (Abcam Inc.,
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Cambridge MA, UK), and polyclonal antibody to human COX-2 (Cayman Chemical, Ann
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Eilati et al. [19] and Hales at al. [16], respectively. Antigen retrieval was performed by
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heating at 98 °C in 10 mM trisodium citrate, pH 6.0, with 0.05% (v/v) Tween-20 for 30 and
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10 min for COX-1 and COX-2, respectively. Endogenous peroxidases were quenched using
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0.3% (v/v) H2O2 in methanol for 15 min. Endogenous biotin was blocked using Avidin/Biotin
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Blocking kit (Vector Laboratories, Burlingame, CA, USA). Sections were incubated with 1%
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(w/v) bovine serum albumin (BSA) (Sigma-Aldrich Japan K.K., Tokyo, Japan) for 1 h,
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followed by monoclonal anti-COX-1 or polyclonal anti-COX-2 antibodies overnight at 4 °C.
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The COX-1 and COX-2 antibodies were diluted in PBS, which contained 1% (w/v) BSA and
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0.1% (v/v) tween-20. The final concentrations of the COX-1 and COX-2 antibody solutions
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were 0.15 and 1 µg/mL, respectively. Normal rabbit IgG (Santa Cruz Biotechnology, Inc.
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Texas, USA) diluted in the same way used for negative control staining. After washing by
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Tris-buffered saline, sections were incubated with biotinylated anti-rabbit IgG (1:200) for 30
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min, followed by incubation with avidin-biotin complex (1:50) (Vector Laboratories) for 30
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min. The immunoreaction products were visualized using a reaction mixture of 0.02% (w/v)
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3, 3´-diaminobenzidine-4HCl and 0.05% (v/v) H2O2, counterstained with hematoxylin, and
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then examined under a light microscope.
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2.4. Western blot
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Mucosal tissues from different segments of the oviduct were homogenized in lysis
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buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 10%
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(v/v) glycerol, 0.1% (w/v) SDS, and 10 mM PMSF) using a POLYTRON® homogenizer
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(Kinematica AG, Tokyo-to, Japan), and then kept on ice for 30 min. They were then
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centrifuged at 12,500 ×g for 15 min at 4 °C, and the supernatant was kept at – 80 °C until use.
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Protein concentration was measured using protein assay reagent (Bio-Rad, Lab., Hercules,
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CA, USA) using BSA for making the standard curve. Samples were mixed with Laemmli
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bromophenol blue and 150 mM Tris-HCl, pH 7.0), and boiled for 10 min. Samples containing
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20 µg of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel
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electrophoresis (SDS-PAGE; 10% separating gel and 4% stacking gel) using 80 V in the
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stacking gel and 120 V in the separating gel.
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After SDS-PAGE, the proteins in the gel were electrophoretically transferred onto a
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0.45 µm Amersham Hybond-C® nitrocellulose membrane at 45 mA for 3 h using a transfer
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buffer (25 mM Tris base, 192 mM glycine and 20% (v/v) methanol, pH 8.3). The membrane
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was incubated with 0.25% (w/v) BSA in washing buffer (20 mM Tris-HCl, pH 7.5, 150 mM
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NaCl, and 0.1% (v/v) tween-20) for 1 h at room temperature to block non-specific binding.
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Membranes were then incubated with monoclonal rabbit anti-human COX-1 (Abcam) or
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monoclonal mouse anti-chicken COX-2 (BD Transduction Lab., Lexington, KY, USA), at
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concentrations of 0.3 µg/mL and 0.25 µg/mL, respectively. Membranes were also incubated
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with anti-chicken β-actin (Santa Cruz Biotechnol., Inc. Texas, USA) diluted at 1:5000, for
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normalization of the density of bands at the final analysis. After washing with the washing
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buffer (10 min x 3 times), the membranes were incubated with peroxidase-labeled secondary
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IgG (GE Healthcare, Buckinghamshire, UK) diluted at 1:2000 in washing buffer for 1 h at
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room temperature. The membranes were washed (10 min × 3 times), and the
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immunoprecipitates on the membrane were treated by Amersham ECLTM Western blotting
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detection reagents (GE Healthcare) for 1 min. The densities of the bands were analyzed using
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an ATTO cooled CCD camera system EZ-Capture II (ATTO Corporation, Tokyo, Japan).
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Optical densities of bands were measured using CS Analyzer® 3.0 software (ATTO, HQ:
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Bunkyoku, Tokyo, Japan).
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2.5. Statistical analysis
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ACCEPTED MANUSCRIPT The significance of differences in gene expression and protein densities among
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different oviductal segments and among different times in an ovulatory cycle was analyzed
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using One-way ANOVA followed by Tukey’s test using IBM SPSS Statistics Desktop 22.0.
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P values of <0.05 were considered significant.
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3. Results
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3.1. COX-1 and COX-2 expression
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Figure 1 shows the expression profiles of COX-1 in different oviductal segments at
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different times after oviposition. The expression level of COX-1 was the highest in the
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infundibulum at all times (Fig. 1A). No significant differences were found among its
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expression levels in the magnum, isthmus, uterus, and vagina at each time point, except at 4 h
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after oviposition when the level was higher in the vagina than in the isthmus (Fig. 1A). As
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shown in Fig. 1B, the COX-1 expression level in the infundibulum was higher at 0 and 24 h
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than at 7 and 16 h after oviposition. In the magnum, COX-1 expression was higher at 24 h
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than at 7 and 16 h after oviposition. In the isthmus, where the expression level was the lowest
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among all oviductal segments, it was higher at 0 h than at 4, 7, and 16 h after oviposition (Fig.
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1B). In the uterus, the expression level was lower at 16 h than the other time points. The
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expression level in the vagina was lower at 16 and 24 h than at 4 h after oviposition (Fig. 1B).
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Figure 2 shows the expression profiles of COX-2 in the different oviductal segments
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at different times after oviposition. The expression level of COX-2 was the highest in the
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uterus relative to the other segments at all times after oviposition except at 4 and 16 h when
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the expression level was not significantly different from that in the vagina (Fig. 2A). Figure
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2B shows the changes in expression level of COX-2 in the different oviductal segments
216
during an ovulatory cycle. The expression level in the infundibulum was higher at 4 h than at
217
24 h after oviposition, whereas there was no difference in the expression levels among
218
different time points in the magnum and isthmus. In the uterus, the COX-2 expression level
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was significantly higher at 24 h than at 4 and 16 h after oviposition. In contrast, the
220
expression level in the vagina was lower at 24 h than at 4 h after oviposition.
221
3.2. COX-1 and COX-2 protein contents Western blot analysis showed that COX-1 and COX-2 proteins existed in the
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different segments of the oviduct (Fig. 3A). Image analysis showed the density of COX-2
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protein was higher in the uterus than in the other segments (data not shown; analysis was
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performed only on the COX-2 bands without normalization by β-actin density). Figure 3B
226
shows the western blot of COX-1 and COX-2 in the uterine mucosa at different times after
227
oviposition. There were no significant differences in density among different times after
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oviposition within COX-1 or COX-2 proteins (Fig. 3C, D).
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3.3. Localization of COX-1 and COX-2
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Immunolocalization of COX-1 in the oviduct at 24 h after oviposition is shown in
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Fig. 4A- E. Immunoreaction for COX-1 was localized in the mucosal epithelium of all
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oviductal segments, tubular gland cells of the uterus, vascular endothelium, leukocytes, and
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smooth muscles in the oviduct at different times after oviposition. Nuclear and cytoplasmic
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staining patterns were frequently demonstrated in those cells (Fig. 4A-E).
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COX-2 immunoreaction signals at 24 h after oviposition are shown in Fig. 5A-E.
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They were mainly localized in the surface epithelium of all segments. Smooth muscles,
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vascular endothelium, and leukocytes in all segments showed positive staining for COX-2.
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Weak staining for COX-2 was found in the tubular gland cells of the uterus (Fig. 5D). Those
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immunoreactions for COX-2 were generally identified in the cytoplasm and around nuclei.
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No staining was observed on the sections incubated with normal rabbit IgG in place of COX-1 or COX-2 antibodies (Fig. 5F).
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well as in the uterine surface epithelium and tubular gland cells isolated by the LCM from the
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uterus at 0 h after oviposition (Fig. 6).
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4. Discussion
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We report here the expression profiles and localization of COXs that are responsible
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for PGs synthesis in the hen oviduct. The major findings are that (1) COX-1 expression was
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the highest in the infundibulum, and was higher around the time of oviposition and ovulation
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(0 and 24 h after oviposition) than the other times; (2) expression of COX-2 was the highest
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in the uterus, and the mRNA expression was the highest immediately prior to oviposition (24
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h after the previous oviposition); and (3) The surface epithelium of all segments was
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immunopositive for both COXs. Asboth et al. [10] showed that PGE2 and PGF2α were
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produced in the hen oviduct and that their amounts were higher in the infundibulum, uterus,
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and vagina than in the magnum and isthmus. The current study supports their report, since the
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expression of COX-1 was the highest in the infundibulum, while COX-2 expression and its
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protein contents were the highest in the uterus followed by the vagina.
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Immunoreactive COX-1 and COX-2 were localized in the surface epithelium,
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capillary endothelial cells, and leukocytes in the lamina propria in all oviductal segments.
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The expression of both COXs in the surface epithelium of the uterus was supported by RT-
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PCR using RNA specifically isolated from those tissues by LCM. In the tubular gland cells of
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the uterus, obvious immunoreaction to COX-1 was identified, but COX-2 showed weak
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immunoreaction. However, the expression of both COX-1 and COX-2 was clearly identified
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in the tubular gland cells isolated by LCM. Thus, the uterine tubular gland cells may have the
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ability to synthesize both COX-1 and COX-2, although the amount of COX-2 protein may be
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small in those cells. Reports on COXs localization in the avian oviduct are not available, but
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studies on the bovine oviduct suggested that COXs regulated its functions by the produced
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ACCEPTED MANUSCRIPT PGs [20]. In the current study, nuclear, perinuclear, and cytoplasmic staining patterns for
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COXs were shown. Similar staining patterns have been shown in the ovary of chicken [16]
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and of other avian species such as quail [17] and ostrich [18]. Subcellular localization of
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COXs is frequently reported to be on the luminal surface of the endoplasmic reticulum and
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the nuclear envelope [21].
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In the infundibulum, COX-1 was identified by western blot and its mRNA
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expression level was higher than in the other segments. Although the changes in the COX-1
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protein density during an ovulatory cycle remain unknown, the expression level was higher at
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0 and 24 h after oviposition. Although reports showing such results in birds are not available,
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there is a report showing that COX-1 mRNA expression in the bovine oviduct was highest
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around ovulation [20]. Thus, we suggest that COX-1 may play a vital role in ovulation by
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regulating the infundibulum, showed an enhanced contractility and close to the ovary around
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ovulation so as to cause ovulation and capture the released ovum [22].
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PGs are important for oviposition in the hens [8, 23], as blocking of their synthesis
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by treatment with indomethacin delayed oviposition [24]. The serum levels of PGs increased
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around the time of oviposition [5] but their source is still controversial. The concentration of
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PGF2α in the uterus showed a peak around the time of oviposition, suggesting a local
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synthesis of PGs [7]. The ruptured ovarian follicles were also reported to play roles for
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oviposition in laying hens [25] by the production of PGs through COX-1 [16]. Contrary to the
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chickens, it was suggested that PGs have no significant roles in neither ovulation nor
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oviposition in the ostrich [18]. The current study shows that COX-1 and COX-2 are
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expressed in the different segments of the oviduct including the uterus, besides that COX-2
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gene expression and its protein density were higher in the uterus than in the other oviductal
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segments. These results suggest that COX-2 may have a role in the regulation of the functions
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of the uterus. It has been reported that COX-2 is inducible by many factors such as
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ACCEPTED MANUSCRIPT inflammatory cytokines [14], mechanical stress [26] or stretching [27]. Thus, such factors
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may control COX-2 expression in the uterus of the laying hen, and affect the local synthesis
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of PGs and oviposition as a consequence, but this may require an additional study to be
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evaluated. Although COX-2 expression in the uterus was up-regulated at 24 h after
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oviposition, the COX-2 protein density did not change during the ovulatory cycle, and the
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reason for this is unknown. Kang et al. [28] mentioned that COX-2 mRNA and protein have
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short half-lives compared with those of COX-1. We assume that COX-2 protein turnover, i.e.,
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replacement of the COX-2 protein with fresh protein, may occur rapidly, and thus, the COX-
300
2 protein density did not significantly change at 24 h after oviposition. The fresh COX-2 may
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be responsible for the increase in PGS synthesis that then influences oviposition in the uterus.
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Monocytes are known to express COXs [29], and our previous study showed that the
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monocyte population in the vagina was higher than in the other oviductal segments [30]. The
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expression level of COX-2 was relatively higher in the vagina until 4 h after oviposition. This
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may be associated with the higher population of leukocytes expressing COX-2 in the vagina
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(Fig. 5E).
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In conclusion, we suggest that the oviductal mucosa synthesizes COXs, providing
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the potential ability to synthesize PGs. COX-1 may have an important role in mediating PGs
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production required for the activity of the infundibulum in engulfing the ovulated ovum.
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COX-2 may have an important role in the production of PGs necessary for performing the
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functions of the uterus including oviposition.
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Acknowledgements
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This work was supported by a Grant-in-Aid for Scientific Research from JSPS (No. 25292161) to Y.Y.
ACCEPTED MANUSCRIPT References
317
[1] Sasanami T, Izumi S, Sakurai N, Hirata T, Mizushima S, Matsuzaki M, et al. A unique
318
mechanism of successful fertilization in a domestic bird. Sci Rep. 2015;5:7700.
319
[2] Wechsung E, Houvenaghel A. The influence of prostaglandins on the in vitro motility of
320
the oviduct of the laying hen. Zentralbl Veterinarmed A. 1975;22:684-8.
321
[3] Lundholm CD. DDE-induced eggshell thinning in birds: effects of p,p'-DDE on the
322
calcium and prostaglandin metabolism of the eggshell gland. Comp Biochem Physiol C
323
Pharmacol Toxicol Endocrinol. 1997;118:113-28.
324
[4] Lundholm CE, Bartonek M. Inhibition of eggshell formation in domestic fowl by
325
indomethacin: relation to calcium and prostaglandin metabolism in the eggshell gland
326
mucosa. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1992;102:379-83.
327
[5] Olson DM, Shimada K, Etches RJ. Prostaglandin concentrations in peripheral plasma and
328
ovarian and uterine plasma and tissue in relation to oviposition in hens. Biol Reprod.
329
1986;35:1140-6.
330
[6] Takahashi T, Tajima H, Nakagawa-Mizuyachi K, Nakayama H, Kawashima M. Changes
331
in prostaglandin F2 alpha receptor bindings in the hen oviduct uterus before and after
332
oviposition. Poult Sci. 2011;90:1767-73.
333
[7] Takahashi T, Ogawa H, Inaba R, Kawashima M. Changes in prostaglandin F
334
concentration in the uterus (shell gland) of the hen oviduct in relation to oviposition and
335
estrogen. Poult Sci. 2004;83:1745-9.
336
[8] Hargrove TL, Ottinger MA. Induced oviposition of precalcified eggs following
337
prostaglandin administration. Poult Sci. 1992;71:548-52.
338
[9] Hertelendy F, Biellier HV. Evidence for a physiological role of prostaglandins in
339
oviposition by the hen. J Reprod Fertil. 1978;53:71-4.
AC C
EP
TE D
M AN U
SC
RI PT
316
ACCEPTED MANUSCRIPT [10] Asboth G, Todd H, Toth M, Hertelendy F. PGE2 binding, synthesis, and distribution in
341
hen oviduct. Am J Physiol. 1985;248:E80-8.
342
[11] Hester PY, Newlon NF, Klingensmith PM. Plasma, follicular, and uterine levels of
343
prostaglandins in chickens laying soft-shelled and shell-less eggs. Poult Sci. 1991;70:1585-93.
344
[12] Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and
345
functions. Physiol Rev. 1999;79:1193-226.
346
[13] Coleman RA, Smith WL, Narumiya S. International Union of Pharmacology
347
classification of prostanoid receptors: properties, distribution, and structure of the receptors
348
and their subtypes. Pharmacol Rev. 1994;46:205-29.
349
[14] Rouzer CA, Marnett LJ. Cyclooxygenases: structural and functional insights. J Lipid Res.
350
2009;50 Suppl:S29-34.
351
[15] Chandrasekharan NV, Simmons DL. The cyclooxygenases. Genome Biol. 2004;5:241-
352
41.
353
[16] Hales DB, Zhuge Y, Lagman JA, Ansenberger K, Mahon C, Barua A, et al.
354
Cyclooxygenases expression and distribution in the normal ovary and their role in ovarian
355
cancer in the domestic hen (Gallus domesticus). Endocrine. 2008;33:235-44.
356
[17] Rodler D, Sinowatz F. Expression of prostaglandin-synthesizing enzymes
357
(cyclooxygenase 1, cyclooxygenase 2) in the ovary of the quail (Coturnix japonica). Folia
358
Biol (Praha). 2015;61:125-33.
359
[18] Rodler D, Sinowatz F. Expression of prostaglandin synthesizing enzymes
360
(cyclooxygenase 1 and cyclooxygenase 2) in the ovary of the ostrich (Struthio camelus). Acta
361
Histochemica. 2015;117:69-75.
362
[19] Eilati E, Hales K, Zhuge Y, Ansenberger Fricano K, Yu R, van Breemen RB, et al.
363
Flaxseed enriched diet-mediated reduction in ovarian cancer severity is correlated to the
364
reduction of prostaglandin E(2) in laying hen ovaries. Prostaglandins Leukot Essent Fatty
365
Acids. 2013;89:179-87.
AC C
EP
TE D
M AN U
SC
RI PT
340
ACCEPTED MANUSCRIPT [20] Odau S, Gabler C, Holder C, Einspanier R. Differential expression of cyclooxygenase 1
367
and cyclooxygenase 2 in the bovine oviduct. J Endocrinol. 2006;191:263-74.
368
[21] Spencer AG, Woods JW, Arakawa T, Singer, II, Smith WL. Subcellular localization of
369
prostaglandin endoperoxide H synthases-1 and -2 by immunoelectron microscopy. J Biol
370
Chem. 1998;273:9886-93.
371
[22] Warren DC, Scott HM. Ovulation in the domestic hen. Science. 1934;80:461-62.
372
[23] Hertelendy F, Yeh M, Biellier HV. Induction of oviposition in the domestic hen by
373
prostaglandins. Gen Comp Endocrinol. 1974;22:529-31.
374
[24] Rzasa J, Paczoska-Eliasiewicz H. Effect of indomethacin and vasotocin on oviposition in
375
the hen (Gallus domesticus). Acta Physiol Pol. 1989;40:111-5.
376
[25] Rothchild I, Fraps RM. On the function of the ruptured ovarian follicle of the domestic
377
fowl. Proc Soc Exp Biol Med. 1944;56:79-82.
378
[26] Fermor B, Weinberg JB, Pisetsky DS, Misukonis MA, Fink C, Guilak F. Induction of
379
cyclooxygenase-2 by mechanical stress through a nitric oxide-regulated pathway.
380
Osteoarthritis Cartilage. 2002;10:792-8.
381
[27] Norregaard R, Jensen BL, Topcu SO, Nielsen SS, Walter S, Djurhuus JC, et al.
382
Cyclooxygenase type 2 is increased in obstructed rat and human ureter and contributes to
383
pelvic pressure increase after obstruction. Kidney Int. 2006;70:872-81.
384
[28] Kang Y-J, Mbonye UR, DeLong CJ, Wada M, Smith WL. Regulation of intracellular
385
cyclooxygenase levels by gene transcription and protein degradation. Prog Lipid Res.
386
2007;46:108-25.
387
[29] Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of
388
prostaglandin synthesis and inhibition. Pharmacol Rev. 2004;56:387-437.
389
[30] Zheng WM, Yoshimura Y. Localization of macrophages in the chicken oviduct: effects
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of age and gonadal steroids. Poult Sci. 1999;78:1014-8.
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Fig. 1. Expression of COX-1 in different segments of oviduct at different times after
393
oviposition. (A) Comparison of COX-1 expression among different segments at each time
394
after oviposition, (B) Changes in COX-1 expression at different times (0 to 24 h) after
395
oviposition within each segment of the oviduct. Values are mean ± SE of the ratio of COX-1
396
to RPS-17 expression (n = 5). Bars with different letters are significantly different (P < 0.05).
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Fig. 2. Expression of COX-2 in different segments of the oviduct at different times after
398
oviposition. (A) Comparison of COX-2 expression among different segments at each time
399
after oviposition, (B) Changes in COX-2 expression at different times (0 to 24 h) after
400
oviposition within each segment of the oviduct. Values are mean ± SE of the ratio of COX-2
401
to RPS-17 expression (n = 5). Bars with different letters are significantly different (P < 0.05).
402
Fig. 3. Western blot of COX-1 and COX-2 in the oviduct of laying hens. (A) Western blot of
403
COX-1 and COX-2 in the mucosa of different oviductal segments at 4 h after oviposition. (B)
404
Western blot of COX-1 and COX-2 in the uterine mucosa at different times after oviposition.
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(C and D) Densities of immunoreaction bands of COX-1 and COX-2 in the uterine mucosa
406
collected at different times (0 to 24 h) after oviposition, respectively. Values are mean ± SE
407
(n = 4) of the ratios of COXs to β-actin (P < 0.05).
408
Fig. 4. Immunostaining for COX-1 in the different segments of the oviduct. (A)
409
Infundibulum, (B) Magnum, (C) Isthmus, (D) Uterus and (E) Vagina at 24 h after oviposition.
410
Immunoreaction products are observed in the cytoplasm and nuclei of surface epithelium (e),
411
uterine tubular glands (g), smooth muscles (m), leukocytes (arrow) and vascular endothelium
412
(arrowhead). Scale bar= 100 µm.
413
Fig. 5. Immunostaining for COX-2 in the different segments of the oviduct. (A)
414
Infundibulum, (B) Magnum, (C) Isthmus, (D) Uterus and (E) Vagina at 24 h after oviposition.
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ACCEPTED MANUSCRIPT Immunoreaction products are observed in the surface epithelium (e), uterine tubular glands
416
(g), smooth muscles (m), leukocytes (arrow), and blood vessels (arrowhead). (F) Negative
417
control section incubated with normal rabbit IgG in place of the primary antibody. Scale bar=
418
100 µm.
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Fig. 6. RT-PCR products of COX-1 and COX-2, in the uterine surface epithelium (E), tubular
420
glands (G) and whole mucosa (M) at 0 h after oviposition. The RNA samples of the surface
421
epithelium and tubular glands were collected using LCM.
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A 10
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Fig. 2. Elhamouly, M. et al. 2
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Fig. 6. Elhamouly, M. et al.
ACCEPTED MANUSCRIPT
We examined the expression and localization of the two isoforms of cyclooxygenase in the different segments of the oviduct of laying hens at different time points in the ovulatory cycle.
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COX-1 and COX-2 are localized in the surface epithelium of all oviductal segments besides the uterine tubular glands.
COX-1 gene expression is the highest at the infundibulum than in the other oviductal
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segments.
COX-2 is expressed significantly higher in the uterus than in the other oviductal
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segments.
COX-1 and COX-2 gene expression may influence the activity of the infundibulum
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during ovulation and the functions of the uterus till oviposition, respectively.