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The expression and cellular localization of galectin-1 and galectin-3 in the Fallopian
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tube are altered in women with tubal ectopic pregnancy
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Junko Nio-Kobayashia, b, Hazirah B. Z. Abidinb, Jeremy K. Brownb, Toshihiko Iwanagaa,
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Andrew W. Horneb, W. Colin Duncanb
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Medicine, Sapporo, JAPAN, bMRC Centre for Reproductive Health, The Queen’s
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Medical Research Institute, The University of Edinburgh, Edinburgh, UK
Laboratory of Histology and Cytology, Hokkaido University Graduate School of
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Short title: Galectins in the Fallopian tube of women
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Correspondence to: Dr. Junko Nio-Kobayashi
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Laboratory of Histology and Cytology, Hokkaido University Graduate School of
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Medicine, Kita 15-Nishi 7, Kita-ku, Sapporo 060-8638, JAPAN. Tel. & Fax: +81-11-
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7067151, E-mail: [email protected]
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Key words: galectin • human Fallopian tube • menstrual cycle • ectopic pregnancy • cilia
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Abstract
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Galectin-1 and galectin-3 are abundantly expressed at implantation sites in the uterus,
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suggesting their involvement in the establishment of pregnancy. In this study, we
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examined the expression and localization of galectin-1 and galectin-3 in the Fallopian
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tubes from non-pregnant women, and in those presenting with tubal ectopic pregnancy.
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There was no significant difference in the expression of both galectin-1 (LGALS1) and
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galectin-3 (LGALS3) transcripts in Fallopian tube across the menstrual cycle. Their
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expression in the Fallopian tube were inversely correlated to each other (r=−0.5134,
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P<0.0001) and differentially localized. Galectin-1 protein was abundant in the stroma of
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non-pregnant Fallopian tubes whereas galectin-3 was mainly localized to the epithelium,
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notably to the cilia of ciliated cells and the apical cytoplasm of secretory cells. In ectopic
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pregnancies, LGALS3 expression was significantly reduced (P<0.0001) but LGALS1
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expression did not alter when compared to non-pregnant Fallopian tube collected during
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the mid-secretory phase. The percentage of Fallopian tube epithelial cells expressing
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galectin-3 in cilia tended to be reduced (P=0.0685) with an accompanying loss of normal
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ciliary structure, while nuclear galectin-3 increased (P<0.05) in ectopic pregnancies.
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Epithelial immunostaining for galectin-1 tended to be elevated in the Fallopian tube from
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women with ectopic pregnancy. Co-culture of human trophoblast-origin SW71 cells
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significantly increased LGALS1 expression in human Fallopian tube epithelial OE-E6/E7
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cells, suggesting that trophoblast-derived products regulate LGALS1 expression in the
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oviductal epithelium. These findings imply a differential contribution of galectin-1 and
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galectin-3 in the homeostasis of human Fallopian tube and in pathophysiology of ectopic
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pregnancy.
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Introduction
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Successful embryonic implantation at the correct site is crucial for human reproduction.
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Ectopic pregnancy is defined as a pregnancy where a blastocyst implants outside the
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normal uterine cavity, and over 95% of ectopic pregnancies are located in the Fallopian
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tube [Walker, 2007; Varma and Gupta, 2012]. Tubal ectopic pregnancy is a life-threating
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condition and is one of the leading causes of maternal death in the first trimester in both
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developed and developing countries [Farquhar, 2005; Varma and Gupta, 2012]. The
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etiology of ectopic pregnancy is still unknown, and early diagnosis and treatment prior to
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the rupture of the Fallopian tube are essential to reduce associated morbidity and mortality.
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Major risk factors for the tubal ectopic pregnancy include tubal damage as a result of
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surgery or infection, cigarette smoking, and in vitro fertilization. These lead to impaired
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tubal transport that delays passage of the embryo along the Fallopian tube and/or to
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alterations in the tubal environment that results in early implantation [Shaw et al., 2010b].
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Galectin is a -galactoside-binding animal lectin which has high affinity for N-
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acetyllactosamine residue (Gal1-3/4GlcNAc) of glycoconjugates [Barondes et al., 1994;
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Hirabayashi et al., 2002]. In mammals, sixteen members of galectin have been identified
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so far and eleven galectin subtypes exist in human tissues (galectin-1 to 4, 7 to 10, 12, 13,
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and 16). Galectins are differentially distributed throughout the mammalian body, and
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involved in various biological functions including cell differentiation, migration, and
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apoptosis, or in pathological events such as inflammation and cancer metastasis [Yang et
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al., 2008]. Galectin-1 and galectin-3 are the major subtypes expressed in the female
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reproductive tracts. They are abundantly expressed in the uterus and at the utero-placental
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interface, and have been implicated in the process of implantation [Powell, 1980;
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Hirabayashi et al., 1984; Poirier et al., 1992; von Wolff et al., 2005)]. As there is
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convincing evidence that galectin-1 and galectin-3 contribute to the establishment of
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successful pregnancy [Blois et al., 2007; Than et al., 2008; Blidner and Rabinovich, 2013;
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Barrientos et al., 2014], we hypothesized that galectins expressed in the Fallopian tube
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may have a role in the pathophysiology of ectopic pregnancy.
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Although the expression and localization of galectins in the uterus and developing
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placenta have been examined in various animals [Phillips et al., 1996; Choe et al., 1997;
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Kim et al., 2008; Yang et al., 2012b; Orazizadeh et al., 2013], only three studies have
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dealt with the expression of galectin-3 in the oviduct/Fallopian tube. Galectin-3 has been
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localized to the epithelium in the Fallopian tube of women [John et al., 2002; Roldán and
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Mrini, 2014], and in the oviduct of cows [Kim et al., 2008] and pigs [Roldán and Mrini,
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2014]. However, there is no information about the detailed expression and localization of
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galectins, particularly galectin-1, in human Fallopian tube across the menstrual cycle, and
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after tubal implantation in ectopic pregnancy. In this study, we investigated the expression
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and cellular localization of galectin-1 and galectin-3 in the Fallopian tube of non-pregnant
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women throughout the menstrual cycle and in the Fallopian tube from women with
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ectopic pregnancy.
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Materials and Methods
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Human Fallopian tube and serum collection
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Ethical approval for this study was obtained from the Lothian Research Ethics Committee,
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and informed written consent was obtained from all of women participating in this study
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(LREC 10/S1102/40). Serum samples (10 mL) and the biopsies (2‒3 cm) at ampullary
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region of the Fallopian tube were collected from study participants at the time of
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hysterectomy for benign gynecological conditions or during surgical management of
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tubal ectopic pregnancy. Women were between 18 and 45 years of age. The stage of the
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menstrual cycle of each patient at the time of hysterectomy was determined by histologic
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examination and staging of an endometrial biopsy taken with the Fallopian tube, and
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measurement of serum estradiol and progesterone levels as described previously [Duncan
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et al., 2011]. Biopsies of non-pregnant Fallopian tubes (n=32) and those from women
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with tubal ectopic pregnancy (n=25) free from trophoblast contamination [Duncan et al.,
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2011] were divided into equivalent portions and either immersed in RNAlater (Ambion,
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TX, USA) at 4°C overnight, and then flash frozen and stored at −80°C for subsequent
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RNA extraction, or fixed in 10% neutral-buffered formalin overnight at 4°C followed by
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storage in 70% ethanol, and subsequent embedding in paraffin wax for
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immunohistochemical staining. Information about the samples used in this study is
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summarized in Table 1 as RNA or paraffin embedded tissue were not available in some
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patients. Serum concentrations of female sex steroids and human chorionic
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gonadotrophin (hCG) were available in only 7 and 15 samples respectively out of the 28
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samples of the Fallopian tube from women with ectopic pregnancy.
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Cell culture
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Human Fallopian tube epithelial cells (OE-E6/E7) [Lee et al., 2001] and human first
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trimester trophoblast cells (SW71) [Straszewski-Chavez et al., 2009] were maintained in
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either DMEM or RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-
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glutamine, 10 unit/mL penicillin, and 0.1 mg/mL streptomycin in 5% CO2 at 37°C. OE-
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E6/E7 cells in DMEM medium were seeded at 1x105 cells per well in 12-well dishes. A
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Transwell® (Corning, Corning, NY, USA) was placed onto each well, and 1x105 SW71
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cells in RPMI 1640 medium were seeded into the insert. Control cells were cultured with
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PRMI 1640 medium without cells in the insert. They were cultured in 5% CO2 at 37°C
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for three days, and OE-E6/E7 cells were collected for RNA extraction.
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Quantitative RT-PCR (qRT-PCR)
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Fallopian tube tissues used for a quantitative gene expression analysis were classified as
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proliferative phase (n=8), mid-secretory phase (n=15), late-secretory to menstrual phase
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(n=8), or ectopic pregnancy (n=25). Total RNA was extracted from frozen human
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Fallopian tube or cultured OE-E6/E7 cells using RNeasy Mini Kit (Qiagen Ltd., Crawley,
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UK) according to the manufacturer’s protocol. RNA (200 ng) was used to prepare cDNA
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by TaqMan Reverse Transcription regents (Applied Biosystems, Foster City, CA, USA).
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The sequences of the primer sets used for this study are described previously [Nio-
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Kobayashi et al., 2014]. Primers were pre-validated by standard PCR and by generating
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standard curves using qRT-PCR. Each reaction buffer contained 5.0 µL 2×PowerSYBR®
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Green PCR Master Mix (Applied Biosystems), 0.5 µL primer pair (5 µM), 3.5 µL of
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nuclease free H2O, and 1.0 µL cDNA, and each reaction was conducted in duplicate. The
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qRT-PCR cycling program consisted of a denaturing step (95°C for 10 min), annealing
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and extension step (95°C for 15 sec and 60°C for 1 min repeated for 40 cycles), and a
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dissociation step (95°C, 60°C, and 95°C for 15 sec each) using a 7900 Sequence Detection
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System (Applied Biosystems). The relative expression levels of each target to the
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housekeeping gene (glucose-6-phosphate dehydrogenase: G6PDH), previously validated
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using geNorm analysis (Primerdesign Ltd, Southampton, UK), were quantified using the
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Δ
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using unpaired t-tests or one-way ANOVA, with pairwise comparison, using GraphPad
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Prism 6 software (GraphPad Software Inc., San Diego, CA, USA), and P<0.05 was
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regarded as significant.
Ct or ΔΔCt methods. After testing for normality, all statistical analyses were performed
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Immunohistochemistry
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Fixed human Fallopian tube tissues collected during the proliferative phase (n=5), the
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mid-secretory phase (n=9), late-secretory to menstrual phase (n=4), and from women with
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ectopic pregnancy (n=26) were available for immunohistochemical analysis. The sections,
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at 5 m thickness, were de-waxed and washed in phosphate-buffered saline (PBS).
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Subsequently the sections were incubated with 3% hydrogen peroxide for 20 min and
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Avidin/Biotin blocking solution (Vector Laboratories Inc., Burlingame, CA) for 15 min
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for each reagent. Then the sections were incubated with normal rabbit or goat serum for
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60 min at room temperature. They were incubated with goat anti-human galectin-1
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antibody (1:1000; AF1152, R&D systems Inc., Minneapolis, MN) or rabbit anti-human
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galectin-3 antibody (1:200; sc-20157, Santa Cruz Biotechnology Inc., Dallas, TX) in
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rabbit or goat serum at 4°C overnight. Control sections were incubated with non-immune
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serum. After washing twice in PBS, the sections were incubated with biotinylated anti-
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goat or anti-rabbit IgG (1:500; Vector laboratories Inc.) for 60 min at room temperature.
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The reaction sites were visualized using Vectastain ABC Elite kit (Vector Laboratories
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Inc.) for 60 min followed by ImmPACTTM DAB Peroxidase Substrate Kit (Vector
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Laboratories Inc.) for 5 min. The sections were counterstained with haematoxylin and
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observed under a light microscope (BX51; Olympus corporation, Tokyo, Japan).
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To quantify the number of cells with positive galectin-3 immunoreaction in either
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the cilia, nucleus, or cytoplasm of the Fallopian tube epithelium collected in the mid-
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secretory phase or during surgery for ectopic pregnancy, at least five images were taken
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from the different parts of the representative sections (n=8 for the mid-secretory phase
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and n=10 for the ectopic pregnancy) using stratified random sampling, and the ratio of
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positive cells in total epithelial cells was calculated by observers blinded to sample source.
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Dual immunohistochemistry
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Some sections after the reaction with anti-galectin-3 antibody overnight were washed
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with PBS and subsequently incubated with AlexaFluor 594-labeled anti-rabbit IgG
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(1:200; Life technologies Japan, Tokyo, Japan) for 2 hours at room temperature. The
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sections were blocked with 10% normal goat serum for 60 min at room temperature and
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then incubated with mouse anti--tubulin antibody (1:2,000; T6793) at 4°C overnight.
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Sections were washed with PBS and incubated with AlexaFluor 488-labeled anti-mouse
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IgG (1:200; Life technologies Japan) for 2 hours at room temperature, and observed under
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a confocal laser scanning microscope (FV300; Olympus, Tokyo, Japan).
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Results
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Expression and localization of galectin-1 and galectin-3 in the Fallopian tube of non-
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pregnant women across the menstrual cycle
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We examined the mRNA expression of galectin-1 (LGALS1) and galectin-3 (LGALS3) by
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qRT-PCR in the Fallopian tube of non-pregnant women during the proliferative, mid-
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secretory, and late-secretory to menstrual phases. The expression of both LGALS1 and
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LGALS3 transcripts did not significantly alter between these phases (Table 1).
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To identify cells expressing galectin-1 and galectin-3 in the Fallopian tube of non-
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pregnant women, we carried out immunohistochemistry using specific antibodies against
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human galectin-1 and galectin-3. There were no clear differences in the staining pattern
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of galectins between the phases. Galectin-1 was predominantly localized to the stromal
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cells, largely fibroblasts and extracellular matrix of the lamina propria, while the
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epithelium showed limited immunoreactivity for galectin-1 (fig. 1A, B). At higher
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magnification, epithelial cells were slightly immunoreactive for galectin-1. However,
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numerous round cells within the epithelium were significantly immunostained for
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galectin-1 (arrowheads in fig. 1B, C).
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In contrast, an intense immunoreactivity for galectin-3 was found in the apical region
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of epithelium while the stroma was weakly immunoreactive for galectin-3 (fig. 1D).
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Galectin-3 in the epithelium was mainly localized to the cilia of ciliated cells (fig. 1E, 1F)
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and the apical cytoplasm of secretory cells (arrows in fig. 1E). Occasionally, the galectin-
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3 immunoreactivity was found in the nucleus or whole cytoplasm of both types of
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epithelial cells in the human Fallopian tube (asterisk in fig. 1F). Dual immunostaining for
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galectin-3 and -tubulin, a maker for cilia, confirmed the localization of galectin-3 both
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in the -tubulin-positive cilia of ciliated cells (fig. 1G-I) and in the apical cytoplasm of
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-tubulin-negative non-ciliated secretory cells (arrows in fig. 1G-I).
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Changes in mRNA expression and localization of galectins in the Fallopian tube from
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women with ectopic pregnancy
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In the Fallopian tube collected after ectopic implantation, the mRNA expression of
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LGALS3 was significantly decreased in the Fallopian tube from women with ectopic
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pregnancy when compared to the Fallopian tube of non-pregnant women during the mid-
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secretory phase (P<0.0001; Table 1). In contrast, the LGALS1 expression did not alter
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between two groups (Table 1).
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Immunohistochemical analysis of the Fallopian tubes from women with ectopic
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implantation revealed that galectin-1 immunoreactivity was abundant in the stroma, as
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was seen in the Fallopian tube from non-pregnant women (fig. 2A). However, the
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epithelium consistently showed elevated immunoreactivity for galectin-1 (fig. 2B) with
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intensified immunolabeling of the round cells within the epithelium (arrowheads in fig.
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2B). Similar to non-pregnant women, galectin-3 immunoreactivity was mainly localized
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to the epithelium (fig. 2C). At higher magnification, epithelial galectin-3 immunoreaction
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was found in the cilia of ciliated cells (fig. 2D) and the apical region of non-ciliated
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secretory cells (arrows in fig. 2D) like the Fallopian tube from non-pregnant women. The
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nuclear immunostaining for galectin-3 was more frequently observed in the Fallopian
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tube from women with ectopic pregnancy (asterisks in fig. 2D). The immunoreactivity
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for galectin-3 in -tubulin-negative secretory cells tended to gather at the plasma
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membrane rather than the apical cytoplasm (arrows in fig. 2D, E).
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Cytoplasmic staining for galectin-3 was abundant in both types of cells in the
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Fallopian tube epithelium with ectopic pregnancy, and ciliated cells containing abundant
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galectin-3 in the cytoplasm appeared to display scattered or irregular ciliary structure
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(arrows in fig. 2F, G). Dual immunostaining for galectin-3 and -tubulin clearly
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demonstrated that cells with abundant immunoreactivity for galectin-3 in the cytoplasm
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had almost lost the -tubulin-positive ciliary structure (arrowheads in fig. 2H-J) while
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cells devoid of the cytoplasmic galectin-3 displayed intact -tubulin-positive ciliary
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structure (arrows in fig. 2H-J). The percentage of cells with galectin-3-positive intact cilia
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tended to decrease in the Fallopian tube from women with ectopic pregnancy (fig. 2K,
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P=0.0685). On the other hand, the number of epithelial cells with nuclear galectin-3
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immunostaining was increased in the Fallopian tube from women with ectopic pregnancy
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(fig. 2L, P<0.01). The number of epithelial cells with cytoplasmic galectin-3
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immunoreactivity in the Fallopian tube did not differ between non-pregnancy and ectopic
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pregnancy (fig. 2M).
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These findings suggest that LGALS3 mRNA expression was significantly decreased
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and alterations in subcellular localization of galectin-3 are remarkable in the epithelial
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cells in the Fallopian tube from women with ectopic pregnancy.
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Galectin-1 and galectin-3 are inversely expressed in the Fallopian tube
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To evaluate the influence of the serum sex steroids on the expression of galectin mRNAs
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in the Fallopian tube, we performed the correlation analysis. Although there was no
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statistical significance, the expression of LGALS1 and LGALS3 seemed to be
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differentially correlated to the concentration of serum sex steroids: LGALS1 expression
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seemed to be positively whereas LGALS3 to be negatively correlated to the concentration
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of both estradiol and progesterone (fig. 3A-D). Interestingly, the mRNA expression of
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LGALS1 and LGALS3 were inversely correlated to each other (r=−0.5134, P<0.0001; fig.
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3E).
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Changes in galectin mRNA expression in human Fallopian tube epithelial OE-E6/E7 cells
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induced by co-culture with trophoblast-origin SW71 cells
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To examine whether the presence of the implanting embryo affects the mRNA expression
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of galectins in human Fallopian tube epithelium, we used two types of cells derived from
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human Fallopian tube epithelium (OE-E6/E7) and human trophoblasts (SW71). OE-
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E6/E7 cells were co-cultured with SW71 cells using transwell system for 3 days, and the
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mRNA expression of LGALS1 and LGALS3 in OE-E6/E7 cells were analyzed by qRT-
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PCR. LGALS1 expression was increased in OE-E6/E7 cells when co-cultured with SW71
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cells (fig. 5A; P<0.0001) while the mRNA abundance of LGALS3 did not alter (fig. 5B).
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This finding suggests that certain molecules secreted from SW71 cells may stimulate the
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mRNA expression of LGALS1 in epithelial OE-E6/E7 cells.
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Discussion
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In this study, we demonstrated the subtype-specific expression and localization of
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galectin-1 and galectin-3 in the Fallopian tube of women. Galectin-1 was mainly localized
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to the stroma but galectin-3 was expressed apical sites of the epithelium in the Fallopian
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tube of non-pregnant women. Although the expression of both galectins did not alter
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during menstrual cycle, the expression of LGALS1 and LGALS3 was inversely correlated
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each other. In the ectopic pregnancies, the expression of LGALS3 was dramatically
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decreased and subcellular localization of galectin-3 changed in association of ciliary loss
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in the Fallopian tube epithelium. On the other hand, the epithelial immunostaining of
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galectin-1 slightly increased in the ectopic pregnancies and co-culture with trophoblast-
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origin SW71 cells significantly enhanced the expression of LGALS1 in the Fallopian tube
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epithelium-derived OE-E6/E7 cells. Although the detailed function of galectins is still
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unclear, these results suggest that galectin-1 and galectin-3 may differentially contributed
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to the pathophysiology of tubal ectopic pregnancy.
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We have for the first time described here the expression and localization of galectin-
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1 in human Fallopian tube. Galectin-1 was predominantly localized to cells in the stroma
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while minimal immunoreactivity was detected in the epithelium. Interestingly,
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intraepithelial round cells were immunostained for galectin-1. We believe that these cells
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are leukocytes as we and other research group have previously identified leukocytes with
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similar morphology in human Fallopian tube epithelium by immunohistochemistry
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[Ulziibat et al., 2006; Shaw et al., 2011]. According to the previous studies, most of
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intraepithelial leukocytes are identified as CD8-positive suppressor T lymphocytes
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[Ulziibat et al., 2006; Shaw et al., 2011]. This suggests a possible contribution of galectin-
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1 in mucosal immunity in human Fallopian tube. Galectin-1 is well-known to regulate
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inflammation [Liu, 2000; Cedeno-Laurent and Dimitroff, 2012] and to be a pivotal
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regulator of the fetomaternal immune tolerance during pregnancy [Blois et al., 2007].
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Previous studies have reported that galectin-3 was localized to the apical surface of
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non-ciliated cells in the oviduct/Fallopian tube of pigs and women [John et al., 2002;
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Roldán and Mrini, 2014], in agreement with our data showing the apical staining of
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galectin-3 in non-ciliated secretory cells of the human Fallopian tube. We further
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demonstrated the unique localization of galectin-3 in the cilia of ciliated cells of human
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Fallopian tube epithelium. Thus it is reasonable to consider differential localization of
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galectin-1 and galectin-3 is established in human Fallopian tube and is related to
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pathophysiology of this organ including ectopic implantation.
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Although the mRNA expression of galectins in the Fallopian tube did not alter during
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menstrual phase, there seems to be weak correlation between the expression of galectins
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in the Fallopian tube and serum concentration of estradiol and progesterone. As shown in
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fig. 3, the expression of LGALS1 tended to be positively correlated to the concentration
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of serum steroids, especially estradiol. On the other hand, the expression of LGALS3
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seemed to be negatively correlated to that. Regulation of the expression of galectin-1 and
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galectin-3 by estradiol and progesterone has been reported in the murine uterus [Choe et
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al., 1997; Hirota et al., 2012], human endometrial epithelial cells [Yang et al., 2012a], and
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human trophoblast cell lines [Yang et al., 2011; Ramhorst et al., 2012]. Although Than et
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al. [2008] noted the existence of estrogen responsive element in the 5’ promoter of
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LGALS1, it remains unclear whether the expression of galectin-3 in the female
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reproductive organ is directly regulated by sex steroid hormones.
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We examined, for the first time, the expression and localization of galectins in the
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Fallopian tube from women with ectopic pregnancy. Unfortunately it is not possible to
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obtain human Fallopian tubes for study during normal early pregnancy, and the effect of
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prolonged exposure to pregnancy hormones cannot be assessed. Thus, we had to compare
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the expression to the Fallopian tube during mid-secretory stage when progesterone levels
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are highest, mimicking the early stages of pregnancy. There was no significant difference
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in the expression of LGALS1 in the Fallopian tube of women with ectopic pregnancy.
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Similar to that in the non-pregnant Fallopian tube, galectin-1 protein was mainly localized
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to the stroma and intraepithelial leukocytes were intensely immunostained for galectin-1,
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while the epithelial galectin-1 immunoreaction was slightly increased in ectopic
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pregnancies. Although we have previously shown that immune cell populations are
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increased in the Fallopian tube from women with ectopic pregnancy [Shaw et al. 2011],
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the number of galectin-1-positive intraepithelial cells did not alter between non-
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pregnancy and ectopic pregnancy (supplementary figure 1), suggesting that there is no
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significant contribution of galectin-1-positive intraepithelial cells to the development of
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ectopic pregnancy. In contrast, there seemed to be an increased epithelial immunolabeling
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more generally in the Fallopian tube from women with ectopic pregnancy. As LGALS1
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mRNA expression in OE-E6/E7 cells was enhanced by co-culture with trophoblast SW71
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cells, the increased galectin-1 immunoreaction in the Fallopian tube epithelium with
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ectopic pregnancy may be a result of ectopic blastocyst implantation and regulated by
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trophoblast products. As sex steroids also can up-regulate galectin-1 in the uterus [Choe
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et al., 1997] and LGALS1 expression tended to be positively correlated to the serum
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concentration of female sex steroids in the Fallopian tube as revealed by this study, the
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prolonged exposure to steroids in early pregnancy is also a potential mechanism for this
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change.
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On the other hand, the mRNA expression of LGALS3 was significantly decreased in
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the Fallopian tube collected from women with ectopic pregnancy compared to that of
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non-pregnant women during mid-secretory phase. This may be partially due to prolonged
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progesterone exposure during ectopic pregnancy. It remains possible that altered galectin-
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3 expression is a cause rather than a consequence of tubal ectopic pregnancy. Down-
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regulation of galectin-3 expression in the mouse endometrium was observed at the
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beginning of pregnancy [Orazizadeh et al., 2013], suggesting that a decreased epithelial
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galectin-3 is associated with successful embryo implantation. However many researchers
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have noted an increased expression of galectin-3 in the uterus during pregnancy, and the
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numbers of implanted embryos decreased when galectin-3 was knocked down selectively
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in mouse endometrium [Yang et al., 2012b]. Therefore the exact role of galectin-3 during
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normal blastocyst implantation and in the Fallopian tube epithelium with ectopic
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pregnancy remains uncertain.
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One interesting observation in the present study was that the subcellular localization
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of galectin-3 changed in the Fallopian tube epithelium from women with ectopic
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pregnancy. Cells with galectin-3-positive cilia significantly decreased in number and
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there was intimate relationship between loss of normal ciliary structure and the nuclear
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or cytoplasmic translocation of galectin-3. Koch et al. [2010] have reported that galectin-
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3 is a novel centrosome-associated protein and knockout of this gene resulted in the
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abnormal morphology of primary cilia in the renal epithelial cells. Recently, the same
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research group demonstrated that galectin-3 at the base of the motile cilia in tracheal
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ciliated cells plays a crucial role in the maintenance of the coordinated orientation and
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stabilization of motile cilia [Clare et al., 2014]. Although the function of galectin-3 in the
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pathogenesis of ectopic pregnancy is still unclear, translocation of galectin-3 from the
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cilia to cell body could be related to ciliary loss and a decrease of ciliary motility, which
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is a major cause of the ectopic implantation [Vasquez et al., 1983]. It remains possible
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that these findings are specific to the Fallopian tubes prone to ectopic implantation, and
396
investigation of the Fallopian tubes damaged by past inflammation would be of interest.
22
397
As galectins are sugar-binding animal lectins, an identification of the ligand
398
glycoconjugates is also important to elucidate the exact role of galectins in the human
399
Fallopian tube. A previous study reported that the binding of biotinylated
400
neoglycoproteins, which contains galectin-recognizing -galactose and lactose, in the
401
epithelium of the oviduct of rabbits [Biermann et al., 1997]. Because sialylation and
402
fucosylation on terminal galactose of glycoconjugates are important modifications on
403
subtype-specific sugar binding affinity of galectins, to investigate the changes in the
404
glycan structures in the human Fallopian tube would be of interest.
405
In conclusion, herein we describe the detailed expression of galectin-1 and galectin-
406
3 in the Fallopian tube from non-pregnant women, and their changes in the Fallopian tube
407
from women with ectopic pregnancy. The differential expression and localization of
408
galectin-1 and galectin-3 suggest the subtype-specific contribution to the homeostasis of
409
human Fallopian tube and to the pathogenesis of ectopic pregnancy.
410
411
412
413
414
23
415
416
417
418
419
420
421
422
Acknowledgements
423
We are grateful to Ms. Lyndsey Boswell, Dr. Fiona Connolly, Ms. Zety Adin, and Ms.
424
Linda Nicol, The University of Edinburgh, for their kind advice and excellent technical
425
support. We thank the research nurses at Royal Infirmary of Edinburgh for help in tissue
426
collection and for all patients participated in this study. We thank Prof. Yeung, The
427
University of Hong Kong, for the use of the human tubal epithelial cell line.
428
429
Funding
430
This study was supported by the Cunningham Trust to WCD. WCD was supported by a
431
Scottish Senior Fellowship from the Scottish Funding Council, AWH by a Clinician
432
Scientist Fellowship from the Medical Research Council, and JN-K by a Postdoctoral
24
433
Fellowship for Research Abroad from Japan Society for the Promotion of Science.
434
435
Conflict of Interest
436
There is nothing to be declared.
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Figure legends
564
Fig. 1. Immunohistochemical images of galectins in the ampullary region of human
565
Fallopian tube from non-pregnant women. The immunoreactivity for galectin-1 is
566
mainly found in the stroma, and the epithelium shows a limited immunoreactivity for
567
galectin-1 (A, B). Round cells within the epithelium are positive in galectin-1
568
immunoreaction (arrowheads in B, C). Galectin-3 is mainly localized in the apical region
569
of the epithelium, and the stroma is weakly immunoreactive for galectin-3 (D). At higher
570
magnification, galectin-3 immunoreactivity is found in the cilia of ciliated cells (E, F)
571
and the apical cytoplasm of secretory cells (arrows in E). Occasionally, the nuclear and
572
cytoplasmic staining for galectin-3 is observed in both types of epithelial cells (asterisk
573
in F shows an example in a ciliated cell). Dual immunostaining for galectin-3 (red) and
574
-tubulin (green) as a maker of cilia clearly demonstrates the positive immunoreaction
575
for galectin-3 in the -tubulin-positive cilia of ciliated cells (G-I) and the apical
576
cytoplasm of -tubulin-negative non-ciliated secretory cells (arrows in G-I). The
32
577
localization of the immunoreactivities did not change between the menstrual phases, and
578
the representative tissue sections at the mid-secretory phase are used for illustration. Insert
579
in A is a control section where the primary antibody was omitted. -tub: -tubulin, G1:
580
galectin-1, G3: galectin-3.
581
Fig. 2. Change in the immunohistochemical localization of galectins in the Fallopian
582
tube of women with ectopic pregnancy. Galectin-1 immunoreactivity is abundant in the
583
stroma of the Fallopian tube from women with ectopic pregnancy (EP) (A) as it is in the
584
Fallopian tube from non-pregnant women. At higher magnification, the immunoreactivity
585
for galectin-1 slightly increased with entire length of epithelium (B). Intraepithelial round
586
cells are also positive in galectin-1 immunroeaction (arrowheads in B). Galectin-3 is
587
localized in both the epithelium and the stroma (C). At higher magnification, the galectin-
588
3 immunoreactivity is found in the cilia of ciliated cells as it is in the non-pregnant women
589
(D) but the nuclear galectin-3 staining in both types of epithelial cells is more frequently
590
observed in the Fallopian tube from women with ectopic implantation (asterisks in D).
591
Galectin-3 immunoreactivity tends to gather at the apical plasma membrane of -tubulin-
592
negative non-ciliated secretory cells (arrows in D and E). Cells with abundant
593
cytoplasmic galectin-3 lose the normal ciliary structure (arrows in F and G). Dual
594
immunostaining for galectin-3 and -tubulin clearly shows the decreased -tubulin-
33
595
immunoreactive ciliary structures in cells with abundant cytoplasmic galectin-3 (arrows
596
in H-J) whereas an intact -tubulin-positive ciliary structure is found in cells without
597
cytoplasmic galectin-3 (arrowheads in H-J). The percentage of epithelial cells with
598
galectin-3-immunoreactive intact cilia tends to decrease in the Fallopian tube with EP (K).
599
On the other hand, the number of cells with nuclear staining for galectin-3 is significantly
600
increased in EP (L). The number of cells with cytoplasmic staining for galectin-3 does
601
not alter between two groups (M). -tub: -tubulin, G1: galectin-1, G3: galectin-3, n.s.:
602
not significant. **P<0.01.
603
604
Fig. 3. The mRNA expression of galectins in the Fallopian tube and serum
605
concentration of female steroids. LGALS1 expression in the Fallopian tube seems to be
606
positively correlated to the serum concentration of estradiol (n=35) and progesterone
607
(n=42) (A, B). On the other hand, the expression of LGALS3 seems to be negatively
608
correlated to these female steroids (C, D). The expression of LGALS1 and LGALS3 in the
609
Fallopian tube is negatively correlated (n=56) (E). n.s.: not significant.
610
611
Fig. 4. Change in mRNA expression of galectins in human Fallopian tube epithelial
612
OE-E6/E7 cells by co-culture with human trophoblast-origin SW71 cells. The mRNA
34
613
expression of LGALS1 is significantly increased in OE-E6/E7 cells co-cultured with
614
SW71 cells (A) while LGLAS3 mRNA expression does not alter (B). Cont: control culture
615
of OE-E6/E7 cells only. ****P<0.0001.
35