- Email: [email protected]
GPR39 is region-specifically expressed in mouse oviduct correlating with the Zn2+ distribution
Accepted Manuscript GPR39 is region-specifically expressed in mouse oviduct correlating with the Zn distribution
2+
Jingqiao Qiao, Huashan Zhao, Ying Zhang, Hongying Peng, Qi Chen, He Zhang, Xueying Zheng, Yaping Jin, Hemin Ni, Enkui Duan, Yong Guo PII:
S0093-691X(16)30459-9
DOI:
10.1016/j.theriogenology.2016.09.040
Reference:
THE 13839
To appear in:
Theriogenology
Received Date: 15 November 2014 Revised Date:
19 September 2016
Accepted Date: 23 September 2016
Please cite this article as: Qiao J, Zhao H, Zhang Y, Peng H, Chen Q, Zhang H, Zheng X, Jin Y, Ni H, 2+ Duan E, Guo Y, GPR39 is region-specifically expressed in mouse oviduct correlating with the Zn distribution, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.09.040. 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.
ACCEPTED MANUSCRIPT 1
GPR39 is region-specifically expressed in mouse oviduct
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correlating with the Zn2+ distribution
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Jingqiao Qiao1#, Huashan Zhao2#, Ying Zhang2, Hongying Peng2, Qi Chen2, He
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Zhang3, Xueying Zheng1, Yaping Jin3, Hemin Ni1, Enkui Duan2*, Yong Guo 1*
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Beijing 102206, China
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College of Animal Science and Technology, Beijing University of Agriculture,
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology,
Chinese Academy of Sciences, Beijing 100101, China
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712100, China
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College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi
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These authors contributed equally to this work.
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*Correspondence authors. Tel. / Fax: 86 10-80799133; 86 10-64807310
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Email: [email protected]; [email protected]
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Abbreviations: GPR39, G-protein coupled receptor 39; GPCR, G-protein coupled
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receptor; NPR, natriuretic peptide receptor; GHS-R, ghrelin receptor; AMG,
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autometallography;
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5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
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DEDTC,
diethyldithiocarbamate;
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X-Gal,
ACCEPTED MANUSCRIPT Abstract G-protein coupled receptor 39 (GPR39) plays a role in cellular and
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physiological processes, including insulin secretion, cell death inhibition, wound
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healing and obesity. Increasing evidence suggests that GPR39 is potently stimulated
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by zinc ions (Zn2+) and is therefore considered a putative Zn2+ receptor. Given the
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importance of Zn2+ in the reproductive system, we proposed that GPR39 might have a
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functional role in the reproductive system. However, the localization of GPR39 in the
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reproductive system remains unknown. Here, we used mice expressing a Gpr39
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promoter-driven LacZ reporter system to detect Gpr39 expression in the reproductive
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system at different phases of the estrous cycle and found an interesting region-specific
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distribution of Gpr39 in the mouse oviduct epithelium, with strong expression at the
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ampulla and weak expression at the isthmus, which was consistent with the results
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using RT-PCR and immunofluorescence. Moreover, using ZnSeAMG staining, we
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found that Zn2+, the putative ligand of GPR39, also showed a distribution similar to
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GPR39 expression, suggesting that their potential interaction mediates fertilization
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and embryo transportation.
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Keywords: GPR39; Oviduct; Estrous cycle; Zinc; Mice
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1. Introduction GPR39 is a G protein-coupled receptor (GPCR) found in all vertebrates [1] and
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expressed in various mammalian tissues, such as digestive system [2], brain [3, 4],
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white adipose tissue [2]. Recent evidence shows GPR39 as an active player in a
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diverse range of physiological and cellular events, such as insulin secretion [5, 6],
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tumorigenesis [7], obesity [8], wound healing [9], cell death inhibition [10], and
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proliferation and differentiation of colonocytes [11]. As an orphan receptor of the
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GPCR family, GPR39 was previously considered as a receptor of obestatin [12-15].
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However, growing evidence shows that GPR39 is potently stimulated by Zn2+ and
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therefore functions as a unique membrane Zn2+ sensing receptor [1, 16]. Although it is
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still unclear whether Zn2+ is the physiological ligand of GPR39 or whether it acts as a
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regulator to synergistically activate GPR39 with other ligands, evidence is
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accumulating that GPR39 plays an important role in mediating Zn2+ signaling in
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various cells and tissue
[17-19].
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In this study, we explore the expression of GPR39 in the female reproductive
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system and the distribution of its putative ligand Zn2+. We used a mouse strain with a
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Gpr39-driven LacZ insertion (Gpr39+/LacZ) that could faithfully monitor Gpr39
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expression to determine GPR39 expression in female reproductive organs. The
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oviduct epithelium showed strong GPR39 expression in the ampulla (the distal end of
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the oviduct) and weak GPR39 expression in the isthmus (the proximal end of the
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oviduct). This pattern of GPR39 expression is similar to the Zn2+ distribution pattern
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along the oviduct.
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2. Materials and methods
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2.1. Animals
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Gpr39+/LacZ
mice,
conventional
whole
body
knockout
via
homologous
recombination, with insertion of the LacZ reporter gene, were generated by Lexicon
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Genetics (Woodlands, TX), as previously described [20]. The adult Gpr39+/LacZ
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(heterozygous), Gpr39+/+ (wild-type) and Gpr39-/- (knockout) mice used in this study
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were cared for as outlined in “The Guidelines for the Care and Use of Animals in
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Research” and maintained in the animal facility of the State Key Laboratory of
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Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences. Mice were
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allowed free access to water and food with a constant photoperiod (12 light: 12 dark).
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Adult female mice in estrus were mated with fertile males at room temperature (25°C),
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in which heterozygous mice were used for breeding. For mouse genotyping, primers
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5’ACCCTCATCTTGGTGTACCT3’ and 5’ATGTAGCGCTCAAAGCTGAG3’ were
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used to amplify a 311-bp band from the wild-type allele, whereas primers
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5’GGAACTCTCACTCGACCTGGG3’
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were used to amplify a 262-bp band from the knockout allele. To separately collect
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samples of normal ampulla and isthmus oviduct, adult CD1 mice (7–8 weeks old)
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were purchased from Vital River Laboratories Co. Ltd. All procedures involving
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experimental animals in this study were in accordance with the Animal Research
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Committee Guidelines of the Institute of Zoology, Chinese Academy of Sciences.
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5’GCAGCGCATCGCCTTCTATC3’
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and
The process of estrus determination was as previously described [21]. The vaginal
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smears from Gpr39+/LacZ mice were smeared onto glass slides. After Giemsa staining,
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the mice at the different phases of the estrus cycle, namely diestrus, proestrus, estrus
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and metestrus, were determined according to the cytological feature.
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2.2. Whole-mount β-galactosidase staining and subsequent tissue sectioning
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The tissues from Gpr39+/+ and Gpr39+/LacZ female mice were dissected with fine
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forceps. Whole tissues were fixed by immersion fixation in 0.2% paraformaldehyde in
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PBS buffer (pH 7.4) containing 2 mmol/L MgCl2 and 5 mmol/L EGTA. Tissues were 4
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mmol/L MgCl2, tissues were transferred into the staining solution (containing 0.1
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mol/L PBS, 2 mmol/L MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mmol/L
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potassium ferricyanide, 5 mmol/L potassium ferrocyanide and 1 mg/mL X-Gal) and
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incubated for 8-10 hours at 37°C. The reaction was stopped by extensive washing in
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PBS, and the tissues were stored in 70% ethanol. After dehydration and paraffin
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embedding,
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deparaffinization and rehydration, the sections were counterstained with eosin.
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2.3. RNA extraction and RT-PCR
tissue
blocks
were
sectioned
(10-µm
thick).
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Samples of ampulla and isthmus oviduct were separately collected, as shown in the
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schematic picture (Fig. 3A). The process for RNA extraction and RT-PCR were as
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previously described [22]. In brief, total RNA from fresh tissues was extracted with
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TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, followed by
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removal of genomic DNA using RNase-free DNase (Promega, Madison, WI). After
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reverse transcription, Gpr39 and Actb were amplified using PCR. PCR reactions were
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conducted in a total volume of 10 µl. Actb was amplified for 23 cycles of denaturation
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at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for
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30 seconds, with a final extension step of 5 minutes at 72°C. The amplified products
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were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide.
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The
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5’TAAAACGCAGCTCAGTAACAGTCCG3’ were used to amplify Actb. To amplify
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Gpr39, the PCR cycle number was adjusted to 30 and 33. The primers were
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5’GCCGGACAGCAAGAAGACAGAC3’ and 5’TACTGCGCCGGGAGGCTAGG3’
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for
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5’CCGGGTTTCACTTCTCGGGC3’ for Gpr39-1b.
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2.4. Immunofluorescence Examination
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primers
Gpr39-1a
5’TGGAATCCTGTGGCATCCATGAAAC3’
and
5’GCCGGACAGCAAGAAGACAGAC3’
and
and
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Frozen sections (20-µm) were fixed in 4% paraformaldehyde for 10 minutes at
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room temperature, rinsed in PBS 3 times, then treated with 0.05% Triton X-100 for 10
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blocked in PBS containing 5% bovine serum albumin (BSA) for 1 hour at 37°C. After
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blocking, the sections were incubated with a rabbit anti-GPR39 antibody (Novus)
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diluted in blocking solution overnight at 4°C in a humid chamber. After 3 washes with
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PBS, the sections were incubated with a secondary antibody (Goat anti-Rabbit
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IgG/FITC; ZF-0311, Beijing Zhong Shan Golden Bridge Biological Technology CO,
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LTD) diluted in blocking solution for 1 hour at 37°C. After another three washes with
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PBS, the nuclei were stained with propidium iodide (PI, sigma) for 10 minutes. The
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sections were observed and photographed using an immunofluorescent microscope
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(Nikon).
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2.5. Zn2+ detection with in vivo selenium method (ZnSeAMG)
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According to ZnSeAMG staining protocols [23, 24], we examined the distribution of
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Zn2+ in different regions of the oviduct. Briefly, 30 mg/kg sodium selenite was
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injected intraperitoneally (IP). After approximately 20 minutes, the animals were
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anesthetized with Avertin followed by transcardial perfusion with 0.9% NaCl and 3%
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glutaraldehyde at room temperature. The oviducts were dissected, placed in 3%
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glutaraldehyde overnight and then in 30% sucrose overnight, both at 4°C. The
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oviducts were then embedded in OCT and sectioned using a cryostat. After drying for
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2 minutes at 55°C, the mounted sections (20-µm thick) were placed in jars filled with
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the Autometallography (AMG) developer and incubated for 2 hours at 26°C. AMG
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development was stopped by replacing the developer with 5% sodium thiosulfate
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solution for 10 minutes. Sections were finally counterstained with toluidine blue. Two
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types of negative controls were prepared: (1) IP injections of diethyldithiocarbamate
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(DEDTC) dissolved in distilled water (1000 mg/kg animal weight) were used to
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specifically neutralize the Zn2+ staining, which was administered 1 hour before
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selenite treatment; and (2) Blank controls, where animals were treated with either no
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selenite or DEDTC.
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3. Results
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3.1. Localization of GPR39 in the female reproductive system of mice
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In the screen for Gpr39 expression in the entire female reproductive system at
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different estrous cycle stages using LacZ staining, we found that Gpr39 was
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specifically expressed in the ampulla of the oviduct (Fig. 1A-D).
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3.2. GPR39 is expressed differentially from ampulla to isthmus in the mouse oviduct Whole-mount oviduct LacZ staining using Gpr39+/LacZ mice further stained blue for
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Gpr39 expression in a region-specific distribution from ampulla to isthmus (Fig. 2A).
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Gpr39-driven LacZ staining was confined to the ampulla epithelial cells of
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Gpr39+/LacZ mice and was absent in Gpr39+/+ mice (Fig. 2B).
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Furthermore, we examined the relative levels of Gpr39 gene transcript isoforms,
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Gpr39-1a and Gpr39-1b [2, 25], in different regions of the oviduct via RT-PCR (Fig.
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3A). Using isoform-specific primers, we found that Gpr39-1a expression was higher
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in the ampulla region and lower in the isthmus region, while Gpr39-1b expression
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was almost absent in the oviduct (Fig. 3B).
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In addition, immunofluorescent staining of ampulla and isthmus epithelia from
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Gpr39+/+ mice showed higher expression of GPR39 protein in ampulla epithelia than
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in isthmus epithelia (Fig. 4A, B). The ampulla and isthmus epithelia of Gpr39-/- mice
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showed no staining (Fig. 4C, D).
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3.3. Zn2+, the putative ligand of GPR39, also presents region-specific distributions in
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mouse oviduct in association with GPR39 expression
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Because Zn2+ is the putative ligand that functionally activates GPR39, we examined
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Zn2+ distribution in the oviduct. Using the ZnSeAMG staining method, we found that
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the mouse oviduct showed different Zn2+ distribution in the ampulla and isthmus
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regions. As shown in Fig. 5A and B, Zn2+ (as depicted by the intensity of dark stain)
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was high in the epithelial layer of the ampulla region but almost absent in the isthmus
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region (Fig. 5E, F). The tunica muscularis also showed a certain degree of staining.
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However, the control group treated with DEDTC, which aimed to neutralize the
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specific signaling of selenite, also showed similar staining in tunica muscularis,
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indicating that it was nonspecific staining (Fig. 5C, G). The blank control groups 7
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(with no selenite) showed no staining at all (Fig. 5D, H).
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4. Discussion The oviduct, also known as a fallopian tube (named after its discoverer, the 16th
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century Italian anatomist Gabriele Falloppio), was previously considered to be a
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conduit that captured the eggs upon their release from the ovarian follicles and
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provided a suitable venue for embryo passage [26, 27]. However, based on
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accumulating evidence, there is now a consensus that the oviduct is a complex organ
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rather than a simple transit zone, with multiple functions in the transport and final
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maturation of gametes, sperm selection, fertilization, early embryo development and
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embryo transportation [28]. These functions of the oviduct are finely regulated by
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ovarian steroids, epithelial secretions, gametes and embryos with which the oviduct
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interacts [29]. The role of the oviduct is also regulated by activating relative receptors,
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such as the β-adrenergic receptor [30], oxytocin receptor [31], LPA receptor [32],
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anandamide receptor [33, 34] and natriuretic peptide receptor (NPR) [35]. The
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region-specific distribution gradient of important receptors/signaling from ampulla to
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isthmus is a hallmark of the oviduct. There have been reports indicating that such
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gradient distribution of Nppa-NPR1 signaling is important for facilitating sperm
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chemotaxis [35] and that anandamide-CB1 signaling is important for embryo
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development and transportation [33, 34]. These gradients within the oviduct comprise
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the fine-tuned local environments that optimize the proper function of gametes and
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early embryo development. In the present study, we have shown direct evidence that
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GPR39 is expressed in specific regions along the oviduct, with strong expression in
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the ampulla epithelium and weak expression in the isthmus epithelium. Moreover, this
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interesting region-specific distribution pattern of GPR39 is in accordance with a
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similar region-specific distribution of Zn2+, the putative ligand of GPR39. These
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results suggest a potential interaction between GPR39 and Zn2+ at the oviduct
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epithelium that might contribute to oviduct function.
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GPR39 was first cloned as a G protein-coupled receptor related to the ghrelin
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receptor (GHS-R) and the neurotensin receptor [36]. As an orphan GPCR receptor, the
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physiological ligand(s) of GPR39 has been of interest to the field. Some research
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groups have identified a Zn2+-binding site on the extracellular domain of GPR39 and 9
ACCEPTED MANUSCRIPT have proposed that Zn2+ could function as a potent agonist of GPR39 in various cell
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types [37, 38], raising the possibility that GPR39 might be the receptor responsible for
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sensing extracellular Zn2+ [39-41], delivering downstream signals and mediating
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cellular responses. GPR39 has been shown to function in Zn2+-induced epithelial
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repair [9] and neuronal cell function [41-43] and as a protector from cell death in a
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hippocampal cell line [10]. Kovacs et al. reported that GPR39 can be activated with
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the dissociation of PKIB in zinc-treated cells [44]. Furthermore, Asraf et al. provided
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evidence that GPR39 possessed Zn2+-dependent activation [19]. Our current study has
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found that GPR39 expression is mainly confined to the distal region of the oviductal
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epithelium, which is the first place where the cumulus-oocyte complex (COC)
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attaches upon entering the oviduct [45].
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In conclusion, we here report that GPR39 shows a region-specific expression
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pattern correlating with Zn2+ distribution along the oviduct. The next functional
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studies using Gpr39-/- mice would reveal exact role in fertilization and embryo
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transportation, especially in the oviduct epithelium, under some stress condition such
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as oxidative stress [10] and chronic restraint stress [46].
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ACKNOWLEDGMENTS We thank Guo’s and Duan’s lab members for comments and suggestion. This work
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was supported by the Strategic Priority Research Program of the Chinese Academy of
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Science XDA04020202-20, the National Natural Science Foundation of China
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(81490742, 31272526, 31200879, 31300957), and the Sciences Knowledge
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Innovation Program of Chinese Academy of Sciences KSCX2-E-W-R-06.
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ACCEPTED MANUSCRIPT FIGURE LEGENDS
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Fig. 1. LacZ staining of the ovary, oviduct (OVD-Ampulla and OVD-Isthmus), uterus,
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cervix, and vagina at different phases of the estrus cycle: diestrus (A), proestrus (B),
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estrus (C) and metestrus (D). The dashed box area is amplified below each row. OVD:
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oviduct. Scale bars: 200 µm
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Fig. 2. Whole-mount LacZ staining showing region-specific distribution of Gpr39
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from ampulla to isthmus in Gpr39+/LacZ female mouse. (A) A blue signal indicates the
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region specific transcription activity of Gpr39 from ampulla to isthmus, compared
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with Gpr39+/+ female mouse. (B) LacZ staining at oviduct sections in Gpr39+/LacZ
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showed that Gpr39 was localized in ampulla epithelial cells. LacZ staining in
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Gpr39+/+ females served as negative controls. Scale bars: 100 µm
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Fig. 3. Differential expression of Gpr39 between ampulla and isthmus. (A) A
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schematic picture showing how different regions of oviduct were separated from
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sample collection. (B) Semi-quantitative RT-PCR results in three independent samples
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showing the relative expression of two Gpr39 isoforms: Gpr39-1a and Gpr39-1b in
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the different regions of oviducts.
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Fig. 4. Immunofluorescence examination of GPR39 in different regions of the oviduct.
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(A, B) Expression of GPR39 in the ampulla (A) and isthmus (B) of Gpr39+/+ mice. (C,
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D) Expression of GPR39 in the oviduct of Gpr39-/- mice. Epi: epithelium. MM:
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muscularis mucosa. Scale bars: 25 µm
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Fig. 5. ZnSeAMG staining showing differential distribution of Zn2+ at ampulla and
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isthmus in oviduct. (A, B) The dark particles indicate Zn2+ localization, which were
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mostly observed in the ampulla epithelium, as marked by red arrows. (E, F) The
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epithelium of the isthmus region showed no such staining. Note that the dark particles
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in the tunica muscularis of both the ampulla and isthmus regions were caused by
26
non-specific staining. (C, G) Non-specific ZnSeAMG staining in the control oviduct
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neutralized using DEDTC. (D, H) ZnSeAMG staining in the blank control oviduct.
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Scale bars: 50 µm
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[22] Chen Q, Zhang Y, Peng H, Lei L, Kuang H, Zhang L, et al. Transient {beta}2-adrenoceptor activation confers pregnancy loss by disrupting embryo spacing at implantation. The Journal of biological chemistry. 2011;286:4349-56.
[23] Gao HL, Feng WY, Li XL, Xu H, Huang L, Wang ZY. Golgi apparatus localization of ZNT7 in the
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[24] Danscher G, Stoltenberg M. Silver enhancement of quantum dots resulting from (1) metabolism of toxic
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zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles. Progress in histochemistry and cytochemistry. 2006;41:57-139.
[25] Rucinski M, Ziolkowska A, Tyczewska M, Malendowicz LK. Expression of prepro-ghrelin and related receptor genes in the rat adrenal gland and evidences that ghrelin exerts a potent stimulating
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effect on corticosterone secretion by cultured rat adrenocortical cells. Peptides. 2009;30:1448-55. [26] Pauerstein CJ, Eddy CA. The role of the oviduct in reproduction; our knowledge and our ignorance. Journal of reproduction and fertility. 1979;55:223-9. [27] Holt WV, Fazeli A. The oviduct as a complex mediator of mammalian sperm function and selection. Molecular reproduction and development. 2010;77:934-43.
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[28] Hunter RH. Components of oviduct physiology in eutherian mammals. Biological reviews of the Cambridge Philosophical Society. 2012;87:244-55. [29] Halter S, Reynaud K, Tahir Z, Thoumire S, Chastant-Maillard S, Saint-Dizier M. [The mammalian oviduct revisited]. Gynecologie, obstetrique & fertilite. 2011;39:625-9.
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[30] Khan I, Omu AE, Fatinikun T, Chandrasekhar B, Kadavil EA, Oriowo MA. Evidence for the presence of beta-3-adrenoceptors mediating relaxation in the human oviduct. Pharmacology. 2005;74:157-62.
[31] Wathes DC, Gilbert CL, Ayad VJ. Interactions between oxytocin, the ovaries, and the reproductive tract in the regulation of fertility in the ewe. Annals of the New York Academy of Sciences. 1993;689:396-410. [32] Shah BH, Catt KJ. Roles of LPA3 and COX-2 in implantation. Trends in endocrinology and metabolism: TEM. 2005;16:397-9. [33] Wang H, Guo Y, Wang D, Kingsley PJ, Marnett LJ, Das SK, et al. Aberrant cannabinoid signaling impairs oviductal transport of embryos. Nature medicine. 2004;10:1074-80. [34] Wang H, Xie H, Guo Y, Zhang H, Takahashi T, Kingsley PJ, et al. Fatty acid amide hydrolase deficiency limits early pregnancy events. The Journal of clinical investigation. 2006;116:2122-31.
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[35] Bian F, Mao G, Guo M, Mao G, Wang J, Li J, et al. Gradients of natriuretic peptide precursor A (NPPA) in oviduct and of natriuretic peptide receptor 1 (NPR1) in spermatozoon are involved in mouse sperm chemotaxis and fertilization. Journal of cellular physiology. 2012;227:2230-9. [36] McKee KK, Tan CP, Palyha OC, Liu J, Feighner SD, Hreniuk DL, et al. Cloning and characterization of two human G protein-coupled receptor genes (GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin receptors. Genomics. 1997;46:426-34. [37] Storjohann L, Holst B, Schwartz TW. Molecular mechanism of Zn2+ agonism in the extracellular
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[38] Storjohann L, Holst B, Schwartz TW. A second disulfide bridge from the N-terminal domain to extracellular loop 2 dampens receptor activity in GPR39. Biochemistry. 2008;47:9198-207.
[39] Hershfinkel M, Moran A, Grossman N, Sekler I. A zinc-sensing receptor triggers the release of intracellular Ca2+ and regulates ion transport. Proceedings of the National Academy of Sciences of the
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[40] Azriel-Tamir H, Sharir H, Schwartz B, Hershfinkel M. Extracellular zinc triggers ERK-dependent activation of Na+/H+ exchange in colonocytes mediated by the zinc-sensing receptor. The Journal of biological chemistry. 2004;279:51804-16.
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[41] Besser L, Chorin E, Sekler I, Silverman WF, Atkin S, Russell JT, et al. Synaptically released zinc triggers metabotropic signaling via a zinc-sensing receptor in the hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2009;29:2890-901. [42] Abramovitch-Dahan C, Asraf H, Bogdanovic M, Sekler I, Bush AI, Hershfinkel M. Amyloid beta attenuates metabotropic zinc sensing receptor, mZnR/GPR39, dependent Ca2+ , ERK1/2 and Clusterin signaling in neurons. Journal of neurochemistry. 2016.
[43] Mlyniec K, Gawel M, Librowski T, Reczynski W, Bystrowska B, Holst B. Investigation of the
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GPR39 zinc receptor following inhibition of monoaminergic neurotransmission and potentialization of glutamatergic neurotransmission. Brain research bulletin. 2015;115:23-9. [44] Kovacs Z, Schacht T, Herrmann AK, Albrecht P, Lefkimmiatis K, Methner A. Protein kinase inhibitor beta enhances the constitutive activity of G-protein-coupled zinc receptor GPR39. The Biochemical journal. 2014;462:125-32.
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[45] Kolle S, Dubielzig S, Reese S, Wehrend A, Konig P, Kummer W. Ciliary transport, gamete interaction, and effects of the early embryo in the oviduct: ex vivo analyses using a new digital videomicroscopic system in the cow. Biology of reproduction. 2009;81:267-74. [46] Ding Q, Li H, Tian X, Shen Z, Wang X, Mo F, et al. Zinc and imipramine reverse the
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ACCEPTED MANUSCRIPT Highlights Using Gpr39+/LacZ mice, we first find Gpr39 is localized in the mouse oviduct.
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GPR39 is expressed differentially from ampulla to isthmus.
3.
Zn2+, the putative ligand of GPR39, also presents region-specific distribution.
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2+
Jingqiao Qiao, Huashan Zhao, Ying Zhang, Hongying Peng, Qi Chen, He Zhang, Xueying Zheng, Yaping Jin, Hemin Ni, Enkui Duan, Yong Guo PII:
S0093-691X(16)30459-9
DOI:
10.1016/j.theriogenology.2016.09.040
Reference:
THE 13839
To appear in:
Theriogenology
Received Date: 15 November 2014 Revised Date:
19 September 2016
Accepted Date: 23 September 2016
Please cite this article as: Qiao J, Zhao H, Zhang Y, Peng H, Chen Q, Zhang H, Zheng X, Jin Y, Ni H, 2+ Duan E, Guo Y, GPR39 is region-specifically expressed in mouse oviduct correlating with the Zn distribution, Theriogenology (2016), doi: 10.1016/j.theriogenology.2016.09.040. 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.
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GPR39 is region-specifically expressed in mouse oviduct
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correlating with the Zn2+ distribution
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Jingqiao Qiao1#, Huashan Zhao2#, Ying Zhang2, Hongying Peng2, Qi Chen2, He
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Zhang3, Xueying Zheng1, Yaping Jin3, Hemin Ni1, Enkui Duan2*, Yong Guo 1*
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Beijing 102206, China
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College of Animal Science and Technology, Beijing University of Agriculture,
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology,
Chinese Academy of Sciences, Beijing 100101, China
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712100, China
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College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi
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#
These authors contributed equally to this work.
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*Correspondence authors. Tel. / Fax: 86 10-80799133; 86 10-64807310
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Email: [email protected]; [email protected]
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Abbreviations: GPR39, G-protein coupled receptor 39; GPCR, G-protein coupled
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receptor; NPR, natriuretic peptide receptor; GHS-R, ghrelin receptor; AMG,
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autometallography;
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5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
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DEDTC,
diethyldithiocarbamate;
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X-Gal,
ACCEPTED MANUSCRIPT Abstract G-protein coupled receptor 39 (GPR39) plays a role in cellular and
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physiological processes, including insulin secretion, cell death inhibition, wound
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healing and obesity. Increasing evidence suggests that GPR39 is potently stimulated
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by zinc ions (Zn2+) and is therefore considered a putative Zn2+ receptor. Given the
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importance of Zn2+ in the reproductive system, we proposed that GPR39 might have a
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functional role in the reproductive system. However, the localization of GPR39 in the
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reproductive system remains unknown. Here, we used mice expressing a Gpr39
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promoter-driven LacZ reporter system to detect Gpr39 expression in the reproductive
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system at different phases of the estrous cycle and found an interesting region-specific
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distribution of Gpr39 in the mouse oviduct epithelium, with strong expression at the
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ampulla and weak expression at the isthmus, which was consistent with the results
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using RT-PCR and immunofluorescence. Moreover, using ZnSeAMG staining, we
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found that Zn2+, the putative ligand of GPR39, also showed a distribution similar to
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GPR39 expression, suggesting that their potential interaction mediates fertilization
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and embryo transportation.
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Keywords: GPR39; Oviduct; Estrous cycle; Zinc; Mice
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1. Introduction GPR39 is a G protein-coupled receptor (GPCR) found in all vertebrates [1] and
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expressed in various mammalian tissues, such as digestive system [2], brain [3, 4],
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white adipose tissue [2]. Recent evidence shows GPR39 as an active player in a
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diverse range of physiological and cellular events, such as insulin secretion [5, 6],
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tumorigenesis [7], obesity [8], wound healing [9], cell death inhibition [10], and
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proliferation and differentiation of colonocytes [11]. As an orphan receptor of the
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GPCR family, GPR39 was previously considered as a receptor of obestatin [12-15].
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However, growing evidence shows that GPR39 is potently stimulated by Zn2+ and
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therefore functions as a unique membrane Zn2+ sensing receptor [1, 16]. Although it is
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still unclear whether Zn2+ is the physiological ligand of GPR39 or whether it acts as a
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regulator to synergistically activate GPR39 with other ligands, evidence is
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accumulating that GPR39 plays an important role in mediating Zn2+ signaling in
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various cells and tissue
[17-19].
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In this study, we explore the expression of GPR39 in the female reproductive
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system and the distribution of its putative ligand Zn2+. We used a mouse strain with a
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Gpr39-driven LacZ insertion (Gpr39+/LacZ) that could faithfully monitor Gpr39
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expression to determine GPR39 expression in female reproductive organs. The
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oviduct epithelium showed strong GPR39 expression in the ampulla (the distal end of
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the oviduct) and weak GPR39 expression in the isthmus (the proximal end of the
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oviduct). This pattern of GPR39 expression is similar to the Zn2+ distribution pattern
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along the oviduct.
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2. Materials and methods
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2.1. Animals
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Gpr39+/LacZ
mice,
conventional
whole
body
knockout
via
homologous
recombination, with insertion of the LacZ reporter gene, were generated by Lexicon
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Genetics (Woodlands, TX), as previously described [20]. The adult Gpr39+/LacZ
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(heterozygous), Gpr39+/+ (wild-type) and Gpr39-/- (knockout) mice used in this study
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were cared for as outlined in “The Guidelines for the Care and Use of Animals in
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Research” and maintained in the animal facility of the State Key Laboratory of
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Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences. Mice were
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allowed free access to water and food with a constant photoperiod (12 light: 12 dark).
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Adult female mice in estrus were mated with fertile males at room temperature (25°C),
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in which heterozygous mice were used for breeding. For mouse genotyping, primers
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5’ACCCTCATCTTGGTGTACCT3’ and 5’ATGTAGCGCTCAAAGCTGAG3’ were
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used to amplify a 311-bp band from the wild-type allele, whereas primers
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5’GGAACTCTCACTCGACCTGGG3’
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were used to amplify a 262-bp band from the knockout allele. To separately collect
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samples of normal ampulla and isthmus oviduct, adult CD1 mice (7–8 weeks old)
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were purchased from Vital River Laboratories Co. Ltd. All procedures involving
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experimental animals in this study were in accordance with the Animal Research
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Committee Guidelines of the Institute of Zoology, Chinese Academy of Sciences.
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5’GCAGCGCATCGCCTTCTATC3’
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The process of estrus determination was as previously described [21]. The vaginal
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smears from Gpr39+/LacZ mice were smeared onto glass slides. After Giemsa staining,
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the mice at the different phases of the estrus cycle, namely diestrus, proestrus, estrus
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and metestrus, were determined according to the cytological feature.
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2.2. Whole-mount β-galactosidase staining and subsequent tissue sectioning
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The tissues from Gpr39+/+ and Gpr39+/LacZ female mice were dissected with fine
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forceps. Whole tissues were fixed by immersion fixation in 0.2% paraformaldehyde in
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PBS buffer (pH 7.4) containing 2 mmol/L MgCl2 and 5 mmol/L EGTA. Tissues were 4
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mmol/L MgCl2, tissues were transferred into the staining solution (containing 0.1
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mol/L PBS, 2 mmol/L MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mmol/L
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potassium ferricyanide, 5 mmol/L potassium ferrocyanide and 1 mg/mL X-Gal) and
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incubated for 8-10 hours at 37°C. The reaction was stopped by extensive washing in
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PBS, and the tissues were stored in 70% ethanol. After dehydration and paraffin
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embedding,
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deparaffinization and rehydration, the sections were counterstained with eosin.
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2.3. RNA extraction and RT-PCR
tissue
blocks
were
sectioned
(10-µm
thick).
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Samples of ampulla and isthmus oviduct were separately collected, as shown in the
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schematic picture (Fig. 3A). The process for RNA extraction and RT-PCR were as
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previously described [22]. In brief, total RNA from fresh tissues was extracted with
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TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, followed by
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removal of genomic DNA using RNase-free DNase (Promega, Madison, WI). After
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reverse transcription, Gpr39 and Actb were amplified using PCR. PCR reactions were
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conducted in a total volume of 10 µl. Actb was amplified for 23 cycles of denaturation
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at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for
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30 seconds, with a final extension step of 5 minutes at 72°C. The amplified products
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were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide.
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The
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5’TAAAACGCAGCTCAGTAACAGTCCG3’ were used to amplify Actb. To amplify
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Gpr39, the PCR cycle number was adjusted to 30 and 33. The primers were
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5’GCCGGACAGCAAGAAGACAGAC3’ and 5’TACTGCGCCGGGAGGCTAGG3’
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for
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5’CCGGGTTTCACTTCTCGGGC3’ for Gpr39-1b.
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2.4. Immunofluorescence Examination
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Gpr39-1a
5’TGGAATCCTGTGGCATCCATGAAAC3’
and
5’GCCGGACAGCAAGAAGACAGAC3’
and
and
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Frozen sections (20-µm) were fixed in 4% paraformaldehyde for 10 minutes at
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room temperature, rinsed in PBS 3 times, then treated with 0.05% Triton X-100 for 10
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blocked in PBS containing 5% bovine serum albumin (BSA) for 1 hour at 37°C. After
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blocking, the sections were incubated with a rabbit anti-GPR39 antibody (Novus)
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diluted in blocking solution overnight at 4°C in a humid chamber. After 3 washes with
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PBS, the sections were incubated with a secondary antibody (Goat anti-Rabbit
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IgG/FITC; ZF-0311, Beijing Zhong Shan Golden Bridge Biological Technology CO,
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LTD) diluted in blocking solution for 1 hour at 37°C. After another three washes with
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PBS, the nuclei were stained with propidium iodide (PI, sigma) for 10 minutes. The
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sections were observed and photographed using an immunofluorescent microscope
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(Nikon).
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2.5. Zn2+ detection with in vivo selenium method (ZnSeAMG)
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According to ZnSeAMG staining protocols [23, 24], we examined the distribution of
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Zn2+ in different regions of the oviduct. Briefly, 30 mg/kg sodium selenite was
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injected intraperitoneally (IP). After approximately 20 minutes, the animals were
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anesthetized with Avertin followed by transcardial perfusion with 0.9% NaCl and 3%
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glutaraldehyde at room temperature. The oviducts were dissected, placed in 3%
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glutaraldehyde overnight and then in 30% sucrose overnight, both at 4°C. The
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oviducts were then embedded in OCT and sectioned using a cryostat. After drying for
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2 minutes at 55°C, the mounted sections (20-µm thick) were placed in jars filled with
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the Autometallography (AMG) developer and incubated for 2 hours at 26°C. AMG
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development was stopped by replacing the developer with 5% sodium thiosulfate
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solution for 10 minutes. Sections were finally counterstained with toluidine blue. Two
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types of negative controls were prepared: (1) IP injections of diethyldithiocarbamate
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(DEDTC) dissolved in distilled water (1000 mg/kg animal weight) were used to
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specifically neutralize the Zn2+ staining, which was administered 1 hour before
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selenite treatment; and (2) Blank controls, where animals were treated with either no
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selenite or DEDTC.
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3. Results
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3.1. Localization of GPR39 in the female reproductive system of mice
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In the screen for Gpr39 expression in the entire female reproductive system at
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different estrous cycle stages using LacZ staining, we found that Gpr39 was
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specifically expressed in the ampulla of the oviduct (Fig. 1A-D).
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3.2. GPR39 is expressed differentially from ampulla to isthmus in the mouse oviduct Whole-mount oviduct LacZ staining using Gpr39+/LacZ mice further stained blue for
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Gpr39 expression in a region-specific distribution from ampulla to isthmus (Fig. 2A).
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Gpr39-driven LacZ staining was confined to the ampulla epithelial cells of
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Gpr39+/LacZ mice and was absent in Gpr39+/+ mice (Fig. 2B).
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Furthermore, we examined the relative levels of Gpr39 gene transcript isoforms,
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Gpr39-1a and Gpr39-1b [2, 25], in different regions of the oviduct via RT-PCR (Fig.
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3A). Using isoform-specific primers, we found that Gpr39-1a expression was higher
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in the ampulla region and lower in the isthmus region, while Gpr39-1b expression
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was almost absent in the oviduct (Fig. 3B).
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In addition, immunofluorescent staining of ampulla and isthmus epithelia from
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Gpr39+/+ mice showed higher expression of GPR39 protein in ampulla epithelia than
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in isthmus epithelia (Fig. 4A, B). The ampulla and isthmus epithelia of Gpr39-/- mice
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showed no staining (Fig. 4C, D).
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3.3. Zn2+, the putative ligand of GPR39, also presents region-specific distributions in
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mouse oviduct in association with GPR39 expression
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Because Zn2+ is the putative ligand that functionally activates GPR39, we examined
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Zn2+ distribution in the oviduct. Using the ZnSeAMG staining method, we found that
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the mouse oviduct showed different Zn2+ distribution in the ampulla and isthmus
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regions. As shown in Fig. 5A and B, Zn2+ (as depicted by the intensity of dark stain)
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was high in the epithelial layer of the ampulla region but almost absent in the isthmus
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region (Fig. 5E, F). The tunica muscularis also showed a certain degree of staining.
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However, the control group treated with DEDTC, which aimed to neutralize the
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specific signaling of selenite, also showed similar staining in tunica muscularis,
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indicating that it was nonspecific staining (Fig. 5C, G). The blank control groups 7
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(with no selenite) showed no staining at all (Fig. 5D, H).
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4. Discussion The oviduct, also known as a fallopian tube (named after its discoverer, the 16th
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century Italian anatomist Gabriele Falloppio), was previously considered to be a
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conduit that captured the eggs upon their release from the ovarian follicles and
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provided a suitable venue for embryo passage [26, 27]. However, based on
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accumulating evidence, there is now a consensus that the oviduct is a complex organ
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rather than a simple transit zone, with multiple functions in the transport and final
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maturation of gametes, sperm selection, fertilization, early embryo development and
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embryo transportation [28]. These functions of the oviduct are finely regulated by
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ovarian steroids, epithelial secretions, gametes and embryos with which the oviduct
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interacts [29]. The role of the oviduct is also regulated by activating relative receptors,
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such as the β-adrenergic receptor [30], oxytocin receptor [31], LPA receptor [32],
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anandamide receptor [33, 34] and natriuretic peptide receptor (NPR) [35]. The
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region-specific distribution gradient of important receptors/signaling from ampulla to
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isthmus is a hallmark of the oviduct. There have been reports indicating that such
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gradient distribution of Nppa-NPR1 signaling is important for facilitating sperm
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chemotaxis [35] and that anandamide-CB1 signaling is important for embryo
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development and transportation [33, 34]. These gradients within the oviduct comprise
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the fine-tuned local environments that optimize the proper function of gametes and
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early embryo development. In the present study, we have shown direct evidence that
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GPR39 is expressed in specific regions along the oviduct, with strong expression in
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the ampulla epithelium and weak expression in the isthmus epithelium. Moreover, this
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interesting region-specific distribution pattern of GPR39 is in accordance with a
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similar region-specific distribution of Zn2+, the putative ligand of GPR39. These
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results suggest a potential interaction between GPR39 and Zn2+ at the oviduct
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epithelium that might contribute to oviduct function.
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GPR39 was first cloned as a G protein-coupled receptor related to the ghrelin
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receptor (GHS-R) and the neurotensin receptor [36]. As an orphan GPCR receptor, the
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physiological ligand(s) of GPR39 has been of interest to the field. Some research
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groups have identified a Zn2+-binding site on the extracellular domain of GPR39 and 9
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types [37, 38], raising the possibility that GPR39 might be the receptor responsible for
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sensing extracellular Zn2+ [39-41], delivering downstream signals and mediating
4
cellular responses. GPR39 has been shown to function in Zn2+-induced epithelial
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repair [9] and neuronal cell function [41-43] and as a protector from cell death in a
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hippocampal cell line [10]. Kovacs et al. reported that GPR39 can be activated with
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the dissociation of PKIB in zinc-treated cells [44]. Furthermore, Asraf et al. provided
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evidence that GPR39 possessed Zn2+-dependent activation [19]. Our current study has
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found that GPR39 expression is mainly confined to the distal region of the oviductal
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epithelium, which is the first place where the cumulus-oocyte complex (COC)
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attaches upon entering the oviduct [45].
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In conclusion, we here report that GPR39 shows a region-specific expression
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pattern correlating with Zn2+ distribution along the oviduct. The next functional
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studies using Gpr39-/- mice would reveal exact role in fertilization and embryo
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transportation, especially in the oviduct epithelium, under some stress condition such
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as oxidative stress [10] and chronic restraint stress [46].
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ACKNOWLEDGMENTS We thank Guo’s and Duan’s lab members for comments and suggestion. This work
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was supported by the Strategic Priority Research Program of the Chinese Academy of
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Science XDA04020202-20, the National Natural Science Foundation of China
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(81490742, 31272526, 31200879, 31300957), and the Sciences Knowledge
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Innovation Program of Chinese Academy of Sciences KSCX2-E-W-R-06.
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Fig. 1. LacZ staining of the ovary, oviduct (OVD-Ampulla and OVD-Isthmus), uterus,
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cervix, and vagina at different phases of the estrus cycle: diestrus (A), proestrus (B),
4
estrus (C) and metestrus (D). The dashed box area is amplified below each row. OVD:
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oviduct. Scale bars: 200 µm
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Fig. 2. Whole-mount LacZ staining showing region-specific distribution of Gpr39
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from ampulla to isthmus in Gpr39+/LacZ female mouse. (A) A blue signal indicates the
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region specific transcription activity of Gpr39 from ampulla to isthmus, compared
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with Gpr39+/+ female mouse. (B) LacZ staining at oviduct sections in Gpr39+/LacZ
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showed that Gpr39 was localized in ampulla epithelial cells. LacZ staining in
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Gpr39+/+ females served as negative controls. Scale bars: 100 µm
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Fig. 3. Differential expression of Gpr39 between ampulla and isthmus. (A) A
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schematic picture showing how different regions of oviduct were separated from
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sample collection. (B) Semi-quantitative RT-PCR results in three independent samples
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showing the relative expression of two Gpr39 isoforms: Gpr39-1a and Gpr39-1b in
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the different regions of oviducts.
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Fig. 4. Immunofluorescence examination of GPR39 in different regions of the oviduct.
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(A, B) Expression of GPR39 in the ampulla (A) and isthmus (B) of Gpr39+/+ mice. (C,
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D) Expression of GPR39 in the oviduct of Gpr39-/- mice. Epi: epithelium. MM:
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muscularis mucosa. Scale bars: 25 µm
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Fig. 5. ZnSeAMG staining showing differential distribution of Zn2+ at ampulla and
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isthmus in oviduct. (A, B) The dark particles indicate Zn2+ localization, which were
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mostly observed in the ampulla epithelium, as marked by red arrows. (E, F) The
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epithelium of the isthmus region showed no such staining. Note that the dark particles
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in the tunica muscularis of both the ampulla and isthmus regions were caused by
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non-specific staining. (C, G) Non-specific ZnSeAMG staining in the control oviduct
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neutralized using DEDTC. (D, H) ZnSeAMG staining in the blank control oviduct.
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Scale bars: 50 µm
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ACCEPTED MANUSCRIPT Highlights Using Gpr39+/LacZ mice, we first find Gpr39 is localized in the mouse oviduct.
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GPR39 is expressed differentially from ampulla to isthmus.
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Zn2+, the putative ligand of GPR39, also presents region-specific distribution.
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1.