Estrogen receptor α is required for oviductal transport of ... · Estrogen receptor a is required...

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THE JOURNAL RESEARCH www.fasebj.org Estrogen receptor a is required for oviductal transport of embryos Shuai Li,* Sofia R. S. ONeill,* Yong Zhang, Michael J. Holtzman, Ken-Ichi Takemaru, Kenneth S. Korach, § and Wipawee Winuthayanon* ,1 *School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA; Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA; Department of Pharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, New York, USA; and § Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA ABSTRACT: Newly fertilized embryos spend the first few days within the oviduct and are transported to the uterus, where they implant onto the uterine wall. An implantation of the embryo before reaching the uterus could result in ectopic pregnancy and lead to maternal death. Estrogen is necessary for embryo transport in mammals; however, the mechanism involved in estrogen-mediated cellular function within the oviduct remains unclear. In this study, we show in mouse models that ciliary length and beat frequency of the oviductal epithelial cells are regulated through estrogen receptor a (ESR1) but not estrogen receptor b (ESR2). Gene profiling indicated that transcripts in the WNT/ b-catenin (WNT/CTNNB1) signaling pathway were regulated by estrogen in mouse oviduct, and inhibition of this pathway in a whole oviduct culture system resulted in a decreased embryo transport distance. However, selective ablation of CTNNB1 from the oviductal ciliated cells did not affect embryo transport, possibly because of a com- pensatory mechanism via intact CTNNB1 in the adjacent secretory cells. In summary, we demonstrated that disruption of estrogen signaling in oviductal epithelial cells alters ciliary function and impairs embryo transport. Therefore, our findings may provide a better understanding of etiology of the ectopic pregnancy that is associated with alteration of estrogen signals. Li, S., ONeill, S. R. S., Zhang, Y., Holtzman, M. J., Takemaru, K.-I., Korach, K. S., Winuthayanon, W. Estrogen receptor a is required for oviductal transport of embryos. FASEB J. 31, 15951607 (2017). www.fasebj.org KEY WORDS: cilia epithelial cells fertility The oviducts (or fallopian tubes in humans) are divided into 3 segments: infundibulum (adjacent to the ovary), ampulla (site of fertilization), and isthmus (connected to the uterus). Each of these segments is made up of epithelial, stromal, and smooth muscle cells. In mammals, the epi- thelial lining of the oviduct consists of secretory and multiciliated cells. The infundibulum and ampulla are composed of more ciliated than secretory cells, whereas the isthmus has more secretory than ciliated cells (1, 2). The multiciliated cells possess motile cilia with a 9+2 arrange- ment of microtubules, composed of a- and b-tubulins. After ovulation, the ovulated oocytes are drawn into the oviduct by the current generated by ciliated cells at the infundibulum (3). The sperm must transit from the uterus through the length of the oviduct to the site of fertilization in the ampulla. The resulting embryos are subsequently transported from the ampulla, through the isthmus, and into the uterus, where implantation takes place. The rela- tive importance of muscular contractility and cilia motility varies along the length of the oviduct. For example, in pigs, isthmus contractions proceed toward and tend to fade beyond the ampulla-isthmic junction (AIJ) (4). In rats and rabbits, inhibition of smooth muscle activity has no effect on embryo transport (57), indicating that ciliary motility is sufficient for transporting the embryos without muscle contractions. Ciliary dysfunction can lead to human infertility or ectopic pregnancy. A rare genetic disorder called primary ciliary dyskinesia (PCD) causes abnormalities in ciliary motility in the ear canal, respiratory tract, and re- productive tract. In men, ciliary dysfunction affects sperm motility (8) whereas, it affects the functionality of the fal- lopian tube in women (911). Some women with PCD are infertile (9, 1113), partly due to fewer cilia per cell and ABBREVIATIONS: Ac a-tubulin, acetylated a-tubulin; AIJ, ampullaisthmic junction; CTNNB1, b-catenin; CBF, ciliary beat frequency; dpc, days postcoitus; ESR, estrogen receptor; IHC, immunohistochemical; IWP2, inhibitor of WNT production 2; KSOM, potassium simplex optimization medium; PCD, primary ciliary dyskinesia; PGR, progesterone receptor; SERM, selective estrogen receptor modulator; SFRP, secreted frizzled- related protein; TBST, Tris-buffered saline-Tween 20; UTJ, uterotubal junction; WNT, wingless-type MMTV integration site family 1 Correspondence: School of Molecular Biosciences, Biotechnology/Life Science BLS202, Washington State University, Pullman, WA 99164, USA. E-mail: [email protected] doi: 10.1096/fj.201601128R 0892-6638/17/0031-1595 © FASEB 1595 Vol.31, No.4 , pp:1595-1607, March, 2017 The FASEB Journal . 134.121.125.9 to IP www.fasebj.org Downloaded from

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THE

JOURNAL • RESEARCH • www.fasebj.org

Estrogen receptor a is required for oviductal transportof embryosShuai Li,* Sofia R. S. O’Neill,* Yong Zhang,† Michael J. Holtzman,† Ken-Ichi Takemaru,‡ Kenneth S. Korach,§

and Wipawee Winuthayanon*,1

*School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA; †Pulmonary andCritical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA; ‡Department ofPharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, New York, USA; and §Reproductive and DevelopmentalBiology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NorthCarolina, USA

ABSTRACT: Newly fertilized embryos spend the first few days within the oviduct and are transported to the uterus,where they implant onto the uterinewall. An implantation of the embryo before reaching the uterus could result inectopicpregnancyand lead tomaternaldeath. Estrogen is necessary for embryo transport inmammals;however, themechanism involved in estrogen-mediated cellular function within the oviduct remains unclear. In this study, weshow inmousemodels that ciliary length and beat frequency of the oviductal epithelial cells are regulated throughestrogen receptor a (ESR1) but not estrogen receptor b (ESR2). Gene profiling indicated that transcripts in theWNT/b-catenin (WNT/CTNNB1) signaling pathway were regulated by estrogen in mouse oviduct, and inhibition of thispathway in a whole oviduct culture system resulted in a decreased embryo transport distance. However, selectiveablation of CTNNB1 from the oviductal ciliated cells did not affect embryo transport, possibly because of a com-pensatorymechanismvia intactCTNNB1 in theadjacent secretory cells. In summary,wedemonstrated thatdisruptionof estrogen signaling in oviductal epithelial cells alters ciliary function and impairs embryo transport. Therefore, ourfindings may provide a better understanding of etiology of the ectopic pregnancy that is associated with alteration ofestrogen signals.—Li, S.,O’Neill, S.R. S., Zhang,Y.,Holtzman,M. J., Takemaru,K.-I.,Korach,K. S.,Winuthayanon,W.Estrogen receptor a is required for oviductal transport of embryos. FASEB J. 31, 1595–1607 (2017). www.fasebj.org

KEY WORDS: cilia • epithelial cells • fertility

The oviducts (or fallopian tubes in humans) are dividedinto 3 segments: infundibulum (adjacent to the ovary),ampulla (site of fertilization), and isthmus (connected totheuterus).Eachof these segments ismadeupofepithelial,stromal, and smooth muscle cells. In mammals, the epi-thelial lining of the oviduct consists of secretory andmulticiliated cells. The infundibulum and ampulla arecomposed of more ciliated than secretory cells, whereasthe isthmushasmore secretory than ciliated cells (1, 2). Themulticiliated cells possess motile cilia with a 9+2 arrange-ment of microtubules, composed of a- and b-tubulins.

After ovulation, the ovulated oocytes are drawn intothe oviduct by the current generated by ciliated cells at theinfundibulum (3). The spermmust transit from the uterusthrough the length of the oviduct to the site of fertilizationin the ampulla. The resulting embryos are subsequentlytransported from the ampulla, through the isthmus, andinto the uterus, where implantation takes place. The rela-tive importance ofmuscular contractility and ciliamotilityvaries along the lengthof theoviduct. For example, inpigs,isthmus contractions proceed toward and tend to fadebeyond the ampulla-isthmic junction (AIJ) (4). In rats andrabbits, inhibition of smooth muscle activity has no effecton embryo transport (5–7), indicating that ciliary motilityis sufficient for transporting the embryos without musclecontractions.

Ciliary dysfunction can lead to human infertility orectopic pregnancy. A rare genetic disorder called primaryciliary dyskinesia (PCD) causes abnormalities in ciliarymotility in the ear canal, respiratory tract, and re-productive tract. Inmen, ciliary dysfunction affects spermmotility (8) whereas, it affects the functionality of the fal-lopian tube in women (9–11). Somewomenwith PCD areinfertile (9, 11–13), partly due to fewer cilia per cell and

ABBREVIATIONS:Ac a-tubulin, acetylated a-tubulin; AIJ, ampulla–isthmicjunction; CTNNB1, b-catenin; CBF, ciliary beat frequency; dpc, dayspostcoitus; ESR, estrogen receptor; IHC, immunohistochemical; IWP2,inhibitor of WNT production 2; KSOM, potassium simplex optimizationmedium; PCD, primary ciliary dyskinesia; PGR, progesterone receptor;SERM, selective estrogen receptor modulator; SFRP, secreted frizzled-related protein; TBST, Tris-buffered saline-Tween 20; UTJ, uterotubaljunction; WNT, wingless-type MMTV integration site family1 Correspondence: School of Molecular Biosciences, Biotechnology/LifeScience BLS202, Washington State University, Pullman, WA 99164, USA.E-mail: [email protected]

doi: 10.1096/fj.201601128R

0892-6638/17/0031-1595 © FASEB 1595 Vol.31,  No.4 , pp:1595-1607, March, 2017The FASEB Journal. 134.121.125.9 to IP www.fasebj.orgDownloaded from

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decreased ciliary beat frequency (CBF) (9, 11, 12). In ad-dition, ectopic pregnancy, a condition where the embryoimplants outside theuterinewall, hasbeenassociatedwithdefective ciliary function of the fallopian tubes (14).Moreover, ectopic pregnancy, because of internal bleedingcaused by rupture of the implantation site, is one of thecauses of maternal death during the first trimester. Theseconditions suggest that the presence of functional ciliatedcells in the fallopian tubes is necessary for normal embryotransport and successful establishment of pregnancy.

The cell morphology of the oviductal epithelium is af-fected by dynamic changes in the ovarian hormones, es-trogen (E2) and progesterone (P4), during the estrous/menstrual cycle inmammals (15, 16). E2 andP4act throughtheir nuclear receptors [estrogen receptor a (ESR1) andestrogen receptor b (ESR2), and progesterone receptor(PGR), respectively]. ESR1 and PGR are expressed in allcell layers of the oviduct (17–19). ESR2 is exclusively de-tected in the cilia of ciliated epithelial cells (20). Previousreports showed that E2 increases the number of ciliatedcells in the mouse neonatal oviduct (17). However, therequirement of ESR1 and -2 for the proper ciliary functionduring embryo transport remains unclear. Moreover, themolecularmechanisms regulated by E2 through ESR1 and-2 in the ciliated epithelial cells during embryo transporthave not yet been explored. In this study, we used globaland conditional Esr-knockout mouse models to addressthe functionality of ESR1 and -2, as well as the possibledownstream signaling cascades that regulate the functionof ciliated cells in the oviduct during embryo transport.

MATERIALS AND METHODS

Mouse models

Wegenerated the conditionaldeletionofEsr1 in theoviductal anduterine epithelial cells using Wnt7aCre/+;Esr1f/f, as previouslypublished (21, 22). Esr1f/f animals were used as control litter-mates. Esr2+/+ and Esr22/2 (or ERb2/2) animals (23) were pur-chased fromTaconic (Germantown,NY,USA).Ctnnb1f/f animalswere obtained from The Jackson Laboratory (Bar Harbor, ME,USA). Ciliated cell-selective cre-expressing animals (or Foxj1Cre+)(24) were crossed with the Ctnnb1f/f animals to generate theconditional deletion ofCtnnb1 (encodingCTNNB1protein) in theciliated cells.

Embryo collection and embryo transfer

Adult female mice (Esr1f/f,Wnt7aCre/+;Esr1f/f, Esr2+/+, Esr22/2,Ctnnb1f/f, and Foxj1Cre+;Ctnnb1f/f) at the age of 8–12 wk weresuperovulated with gonadotropins [5 U of pregnant mare’s se-rum gonadotropin and 5 U of human chorionic gonadotropin(hCG); EMD–Millipore, Billerica, MA, USA] (22). Immediatelyafter hCG injections, the females were housed with wild-typemale breeders overnight, and the copulatoryplugswere checkedthe following morning and designated as 0.5 d postcoitus (dpc).At 3.5 dpc (10 AM), the female reproductive tracts were dissectedfrom the animals. The embryos were first flushed from the uteri,then the oviducts. The number of embryos from each region(uterus vs. oviduct) was calculated as a percentage of the totalmorula and blastocyst stage embryos collected. Only the viableembryos were included in the experiment. Nonviable embryos,such as empty zona pellucida, unfertilized eggs, and fragmented

eggs, were excluded, because we could not distinguish betweenthe previously ovulated eggs and superovulated eggs that werenot fertilized.

For embryo transfer, 1-cell-stage embryos (zygotes) werecollected from CF-1 donor mice and transferred into the oviductof Esr1f/f and Wnt7aCre/+;Esr1f/f pseudopregnant females thatwere mated with vasectomized males (22). The embryos werecollected at 3.5 dpc from the oviduct and uterus, and only viableembryos were included in the experiment. Nonviable embryoswere excluded from the experiment, because they could not bedistinguished accurately from ovulated, unfertilized eggs gen-erated by the pseudopregnant recipients.

Immunohistochemical andimmunofluorescent analysis

Another set of animals was bred with wild-type males, and theembryos were left intact without flushing for histologic analysis.The oviducts were collected at 0.5 or 3.5 dpc and fixed in 10%buffered formalin solution. The uteruswas collected fromEsr2+/+

and Esr22/2 females at 3.5 dpc. Then, the tissues were embed-ded in paraffin and cut at 5 mm. The tissue sections weredeparaffinized and incubated with citrate buffer (pH 6.0), byusing the heat-induced epitope-retrieval method. The tissueswere blocked with normal horse serum and incubated with pri-mary antibodies against acetylated a-tubulin (Ac a-tubulin, 1:6000, T6793; Sigma-Aldrich, St. Louis, MO, USA) and CTNNB1(1:200, 610154; BDBiosciences, San Jose, CA,USA). Thedetectionof ESR1, ESR2, and Ki67 was performed as previously reported(25). DAB chromogen was used as an antigen labeling system(Dako, Carpentaria, CA, USA). The tissues were mounted andcoverslipped. Hematoxylin and eosin (H&E) staining was per-formed according to standard histologic procedure. The imageswere obtained with the DMi8 microscope (Leica, Bannockburn,IL, USA). In some experiments, expression of Ac-a-tubulin wasevaluatedwithdonkey anti-mouse 488 (ThermoFisher Scientific,Waltham, MA, USA) as a secondary antibody, and the tissueswere coverslippedwithProlongGoldwithDAPI (ThermoFisherScientific). The fluorescent images were taken with a UV 510microscope (Zeiss, Thornwood, NY, USA).

Quantification of Ki67-positive cells

The oviduct tissues stained with Ki67 antibody were imaged at3100 magnification. Individual images were imported into Fijisoftware (26). The numbers of epithelial cells and Ki67-positivecells were counted using the “Cell-counter” plug in. The Ki67-positive cells were calculated and presented as a percentage ofpositive cells per total epithelial cells in that microscopic field. Atleast 5 areas (images) were used from each animal for cellcounting (n = 4–5 animals per group).

Ciliary length measurement

Formalin-fixed oviductal sections from 4 animals per genotypewere stainedwithAca-tubulin, as described above. Imagesweretaken at the infundibulum and ampullary regions with a LeicaDMi8 microscope with a 3100 objective lens. The scale bars(10mm)were automatically embedded into all images for furtherciliary length analysis in Fiji software. A total of 51, 82, 87, and 96images were obtained of the Esr1f/f infundibulum, Esr1f/f am-pulla, Wnt7aCre/+;Esr1f/f infundibulum, and Wnt7aCre/+;Esr1f/f

ampulla, respectively. The observer was blinded to the animal’sgenotype tominimize bias. To calibrate the length of 10-mmscalebars, the “straight-line” tool was used to draw a line and set to aknown distance of 10 mm using the “Set Scale” tool. Then, theciliary length of each ciliated epithelial cell was measured

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from the apical membrane (base of the cilia) to the tip of thecilia using the “Straight-line” tool. Using all images, 674, 1446,1098, and 1540 ciliated epithelial cells were measured for cil-iary length analysis from Esr1f/f infundibulum, Esr1f/f am-pulla, Wnt7aCre/+;Esr1f/f infundibulum, and Wnt7aCre/+;Esr1f/f ampulla, respectively.

Microarray analysis

The oviducts were collected from Esr1f/f and Wnt7aCre/+;Esr1f/f

females at 0.5 dpc (n = 4 per genotype). RNA extraction andmicroarray analysis were previously described (22). The datawere deposited in the National Center for BiotechnologyInformation (Bethesda,MD,USA)Gene ExpressionOmnibus(Accession No. GSE37471). In brief, the microarray dataset wasanalyzed using Partek Genomic Suite software (Partek, St. Louis,MO,USA).Genes thatwereexpressed$|1.5|-fold inWnt7aCre/+;Esr1f/f compared to Esr1f/f oviducts were considered signif-icantly different. Differentially expressed genes were ana-lyzed in Ingenuity Pathway Analysis (Qiagen, Valencia, CA,USA).

Oviduct collection for gene expression analysis

Adult female mice (8–12 wk old) were used for the gene ex-pression analysis of WNT/CTNNB1 signaling pathway. Theestrous cycle was determined by vaginal cytology, as previouslyindicated (27). The oviducts were collected at each stage of es-trous cycle, and all regions of the oviduct (infundibulum, am-pulla, and isthmus) were dissected and collected separately.RNA was extracted from the dissected tissues using Trizol re-agent (ThermoFisher Scientific), according to themanufacturer’sprotocol. RNA from each sample (1 mg) was incubated withDNaseI to eliminate the DNA in the samples, and the sampleswere reverse transcribed to cDNA with SuperScriptII (ThermoFisher Scientific). Then, the cDNAs were used as a template forreal-time PCR analysis. Quantitative PCR was performed withFastSBYRGreenmastermix (ThermoFisher Scientific) accordingto the manufacturer’s protocol. The expression values werenormalized to ribosomal protein L7 (Rpl7) and calculated aspreviously described (21). The primer sequences are (F, forward;R, reverse): Ctnnb1 (F: 59-TCCGAGGACTCAATACCATTCC-39and R: 59-AGGACCCCTGCAGCTACTCTT-39); Cby1 (F: 59-AGACAGGAGACGGAAGTGCT-39 and R: 59-GCTGAATAT-GCTGCCAAAGA-39); Sfrp2 (F: 59-TCCTGCCGGCCACAGA-39and R: 59-GTTGTCGTCCTCATTCTTGGTTT-39), Axin2 (F:59-GTCACAGGATGTCTGGCAGT-39 and R: 59-TTATGCTT-TGGGCACTATGG-39); Cdh1 (F: 59-CTGGGCAGAGTGAG-ATTTGA-39 and R: 59-CCACTTTGAATCGGGAGTCT-39);Fgf18 (F: 59-TTCTACTGCTGTGCTTCCAGGTT-39 and R:59-ACGTGGATGCGGAAGTCC-39); Foxj1 (F: 59-CTCTGAGC-CAGGCCTCACATT-39 and R: 59-GGTCAACATCCA-CAGGCTGA-39); Fzd9 (F: 59-GGGATGGCCAGCTCACTTT-39and R: 59-CCTCGTGGCCCCATTTTT-39); Fzd10 (F: 59-GAA-GCCAACAGCAGCTACTTTCA-39 and R: 59-GCCACCCT-GCGCATCA-39);Kif3a (F: 59-GACATCAGTGGGTCAGAGGA-39and R: 59-TTTACCTGCTTGGTCCCTTC-39); Kif6 (F: 59-GGAC-TCGGAAAGGTCAAGGG-39 and R: 59-AGTAAATCCCTTGC-TGCTGCT-39); Kif9 (F: 59-CCGGTGGCGTTAGCTAAAAT-39and R: 59-CCCTCTCCGAGTGTCCTTCT-39); Kif19 (F: 59-TCT-ATGTTCGGACCCTCAATGA-39 and R: 59-ATCACCCCTTT-GGAGTCTTCC-39);Tcf3 (F:59-GCTGTCACAGACACAGTCTCAGC-39and R: 59-TGAATGCATGGCTTCTTGCT-39); Wnt2 (F:59-AACACCCTGGACAGAGATCACA-39 and R: 59-GCGTAA-ACAAAGGCCGATTC-39); Wnt7a (F: 59-CTTTCTCAGCC-TGGGCATAGTCT-39AandR:ATGATGCTCGCACCCAGAG-39);and Wnt7b (F: 59-AACCTTCACAACAATGAGGCG-39 and R:59-CACACCGTGACACTTACATTCCA-39).

Immunoblot analysis

Another set of animals was bred with wild-type males (3animals/genotype). The whole oviducts were collected at 0.5dpc, snap frozen, and stored at280°C until use. The proteinwasextracted from theoviductswith tissueprotein extraction reagent(Thermo Fisher Scientific) with Halt protease and phosphataseinhibitor cocktail. A total of 2 mg protein/lane was loaded into10% SDS-polyacrylamide gels. The protein was transferred ontonitrocellulose membranes using the Trans-Blot Turbo TransferSystem (Bio-Rad Laboratories, Hercules, CA, USA). The mem-branes were blocked with 5% nonfat dry milk in Tris-bufferedsaline with Tween 20 (5% milk TBST) and incubated at 4°Covernight with the following primary antibodies in 5% milkTBST: Ac a-tubulin (TUBA1B, 1:1000, T6793; Sigma-Aldrich),ADP-ribosylation factor-likeprotein13B (ARL13B,1:1000, 17711-1-AP, Proteintech), b-tubulin IV (TUBB4, 1:1000, MU178-UC,BioGenex, Fremont, CA, USA), and b-actin (1:5000, SC-47778,Santa Cruz Biotechnology, Dallas, TX, USA). Then, the mem-branes were washed 3 times for 10 min each in TBST andincubated with horseradish-peroxidase conjugated goat anti-mouse (Santa Cruz Biotechnology) or goat anti-rabbit (Abcam,Cambridge,MA,USA)dilutedat 1:5000 in 5%milkTBSTat roomtemperature for 1 h andwashed 3 times for 10min each in TBST.Chemiluminescence detection was performed with ECL clarity(Bio-Rad) and detected with ChemiDoc MP System (Bio-Rad).The band intensities were quantified and normalized to b-actinusing Fiji software with Analyze and Gel Tools.

Human fallopian tube tissues

Human fallopian tubes were surgically removed from 3 womenwho underwent gynecologic procedures (such as tubal ligation,salpingohysterectomy, or oosalpingohysterectomy) at the StonyBrook University School of Medicine. The tissues were collectedas biopsies or discarded tissues, fixed with formalin, and em-bedded in paraffin. The paraffin-embedded tissue blockswere inthearchival collectionsof theStonyBrookUniversity’sPathologyDepartment and were deidentified and shipped to WashingtonState University. The ethics approvals for this study wereobtained from both Washington State University and StonyBrook University Medical Center. The expression analysis ofCTNNB1 and Ac a-tubulin was performed with immunohisto-chemical analysis as indicated above.

Ex vivo oviduct culture

The procedures for ex vivo culture of the mouse oviduct wasadapted from a previous report (28). In brief, the oviducts weredissected from female mice at 10–11 AM of 0.5 dpc. Then theoviducts were washed in Leibovitz-15 (Thermo Fisher Scientific)with 1% fetal bovine serum (Gemini Bio-Products, West Sacra-mento, CA, USA) and trimmed of gonadal fat. The mesentericligaments were carefully cut with fine-point dissecting forcepsand detached from the oviduct to linearize (uncoil) the oviductunder a heated stage stereomicroscope (LeicaMicrosystems, Inc.,BuffaloGrove, IL,USA) (29).Uncoiling theoviducts at the start ofculture allowed us to accurately locate the embryos at the end ofthe experiment without additional oviduct manipulations thatcould affect embryo location. The uncoiled oviducts with em-bryos inside were placed on a Transwell insert (Corning, Inc.,Corning, NY, USA) in a 24-well plate with potassium simplexoptimization medium (KSOM), which is suitable for embryodevelopment, at 37°Cwith5%CO2.Theoviduct fromthe left sidewas cultured with 0.1% DMSO (Sigma-Aldrich) in KSOM andthe right side of the oviduct from the same animal was cul-turedwith 5mMinhibitor ofWNTproduction 2 (IWP2; Tocris,Bristol, United Kingdom) diluted in 0.1% DMSO in KSOM.

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The medium containing DMSO or IWP2 was refreshed every24 h. After 3 d, the oviducts were placed on a slide, and thelocation of the embryos was determined. Embryo transportwas calculatedwith adissectingmicroscope equippedwith aneyepiece micrometer, by measuring from the AIJ to the far-thest embryo in the oviduct toward the uterotubal junction(UTJ). The distance of the farthest embryo was used becausemost embryos travel singly, not as a group, within the ovi-ductal lumen. After the location of the embryoswasmeasuredin the oviducts, the embryos were flushed to evaluate the ef-fect of the treatment on embryo development. The oviductswere also collected for RNA expression analysis ofAxin2. Theexperiments were repeated twice from a total of 4–5 animalsper treatment.

Measurement of CBF

Toevaluate theCBF inEsr1f/f andWnt7aCre/+;Esr1f/f females,wemeasured the beat frequency in the ex vivo oviducts collected at0.5 dpc (n = 3 per genotype). The oviducts were openedlongitudinally. The infundibulum and isthmus were mounted(leaving the ampulla unmounted), with tissue adhesive onto aglass-bottom dish. During imaging, the oviduct tissue was cul-tured in 4mlLeibovitz-15 at 37°Cwith aheated chamber. Imageswere recorded from the ampulla at 100 frames per second for 10 swith a high-speed camera (Andor Zyla, Andor, Concord, MA,USA)with a3100 objective connected to an inverted bright-fieldmicroscope (DMi8, Leica). The regions of interest (ROIs) at 434pixels were selected from the sample at the tip of the cilia. Thelight signal intensities from each ROI over time were collectedusing Fiji software and transformed into a frequency using a fastFourier transform algorithm in AutoSignal software (SystatSoftware Inc., San Jose,CA,USA). The total of 168–469ROIswererecorded from each animal.

Statistical analysis

Results reached statistical significance at P , 0.05 using anunpaired Student’s t test (GraphPad Prism, La Jolla, CA,USA) for comparison of 2 separated groups of treatment or

genotype. The Mann-Whitney U test was used to separatemedians for the CBF data. Comparison of gene expressionsduring estrous cycle was made using 1-way ANOVA, Tukeypost hoc test.

RESULTS

ESR2 localizes to cilia in the oviductepithelium but is dispensable for theembryo transport function of the oviduct

To first confirm whether ESR1 and -2 are expressed inthe ciliated epithelial cells in mice, we assessed the lo-calization of ESR1 and -2 proteins in comparison withAc a-tubulin, a cilia marker within the oviduct. Weobserved a positive staining of ESR1 in the cell body ofall oviductal cell layers, including epithelial, stromal,andmuscle cells, whereas ESR2was only detected inAca-tubulin-positive ciliary stalks of ciliated epithelialcells (at the infundibulum and ampulla region; Fig. 1A).

Based on the high abundance of ESR2 protein specifi-cally in the cilia of oviductal ciliated cells,wehypothesizedthat ESR2 has a role in the formation and function of ciliaand is therefore critical for the embryo transport in theoviduct. To investigate the functional requirement of ESR2during the embryo transport, we determined the numberof embryos present in theuterus, theoviduct, or both at 3.5dpc of Esr22/2 mice. Although the number of embryoswas slightly lower in Esr22/2 because of an ovulatorydefect (30), therewasnosignificantdifferencebetween thenumber of morulae and blastocysts in Esr22/2 whencompared toEsr2+/+ females (Fig. 1B).Almost 100%of theembryos were retrieved from the uterus at 3.5 dpc fromthe Esr2+/+ and Esr22/2 animals (Fig. 1C). Uterine cross-sections indicated that the embryos were present inthe uterine lumen in both Esr2+/+ and Esr22/2 animals at3.5 dpc (Fig. 1D). These findings indicate that ESR2 is

Figure 1. ESR2 is exclusively present in the cilia of oviductal epithelial cells but is not necessary for embryo transport. A)Immunofluorescent staining of Ac a-tubulin and IHC analysis of ESR1 and -2 of wild-type mouse oviduct in the infundibulum,ampulla, and isthmus. Insets are negative controls. Note that multiciliated cells (indicated by Ac a-tubulin IHC) were moreabundant in the infundibulum and ampulla compared to isthmus. B) Number of embryos at morulae and blastocyst stagescollected from the oviduct and the uterus of control, Esr2+/+ (n = 3 mice), and Esr22/2 (n = 4 mice) females at 3.5 dpc. C)Percentage of the embryos retrieved from the uterus in Esr2+/+ and Esr22/2 females at 3.5 dpc. Graphs represent means 6 SEM.D) H&E staining of embryos (E) within the uterine lumens of Esr2+/+ and Esr22/2 females at 3.5 dpc.

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dispensable for the early development and transport ofembryos.

Epithelial ESR1 is necessary for the embryotransport in the oviduct

Because it has been established that E2 is necessary for thetransport of embryos in murine models (31) and we ob-served no effect of Esr2 ablation on the embryo transport,we hypothesized that the E2-dependent embryo transportoccurs through ESR1, specifically epithelial ESR1. Un-fortunately, the global Esr12/2 females are infertile andthe hormonal profile (elevated testosterone, E2 and lutei-nizing hormone) is abnormal. Therefore, the Esr12/2

model is not suitable as a tool to study embryo transportunder the influence of E2. To circumvent this, we haverecently generated a conditional-knockout mouse modelwhere Esr1 is selectively ablated only in the epithelial cells(Wnt7aCre/+;Esr1f/f animals) of the female reproductivetract, including the oviduct and uterus (21, 22). Moreover,these females showed normal features of estrous cyclicityand ovulatory response (21). We confirmed that ESR1proteinwasnot present in the epithelial cells of the oviductinWnt7aCre/+;Esr1f/f mice butwas detected in Esr1f/f mice(control littermates) (Fig. 2A).Wealso previously reportedthat embryos in Wnt7aCre/+;Esr1f/f females die beforethe 2-cell stage (22). Therefore, to assess whether the em-bryo transport function of the oviduct was dysfunctional,we transferred 1-cell stage embryos (zygotes) from wild-type donors into the oviducts of pseudopregnant Esr1f/f

or Wnt7aCre/+;Esr1f/f animals and retrieved the embryosat 3.5 dpc. The majority of the embryos (82%) retrievedfrom Esr1f/f females were located in the uterus, whereas100% of the embryos retrieved from Wnt7aCre/+;Esr1f/f

females were retained only in the oviducts—none in theuterus (Fig. 2B). These results suggest that E2-dependentembryo transport function in the oviduct is mediatedthrough the epithelial ESR1.

E2-induced proliferation of epithelial cells in the femalereproductive tract is mediated by ESR1 (21). To evaluatewhether a lack of epithelial Esr1 causes aberrant epithelialcell proliferation in Wnt7aCre/+;Esr1f/f females and con-tributes to defective embryo transport, we examined theproliferative indexof these cells usingKi67 IHCanalysis inEsr1f/f andWnt7aCre/+;Esr1f/f oviducts at 0.5 dpc (Fig. 2C).There was no indication of proliferation differences in theWnt7aCre/+;Esr1f/f oviducts, because a comparable num-ber of Ki67-positive epithelial cells was observed in Esr1f/f

and Wnt7aCre/+;Esr1f/f oviducts (Fig. 2D). To next de-terminewhether the loss of epithelial ESR1 impacts ciliarymorphology, we evaluated the presence of cilia in theoviduct using H&E staining with high-magnification im-aging (Fig. 2E) and the presence of Ac a-tubulin (Fig. 2F).We found that the positive staining of Ac a-tubulinwas comparable between Esr1f/f and Wnt7aCre/+;Esr1f/f

oviducts (Fig. 2F). Moreover, the levels of ciliary proteins(Ac a-tubulin, ARL13B (32), and TUBB4 (33)) were notsignificantly different between Esr1f/f and Wnt7aCre/+;Esr1f/f oviducts (Fig. 2G, H). In addition to overall ciliarymorphologyandprotein levels,weassessedthe lengthof thecilia stalk, because a previous finding indicated that normal

ciliary length is crucial for cilia function in the oviduct (34).We found that the ciliary length at the infundibulum, mea-sured from the oviductal tissues stained with Ac a-tubulin,was significantly longer in theWnt7aCre/+;Esr1f/f comparedwith the Esr1f/f oviducts (Fig. 2I). The ciliary length in theampulla region had a tendency to be longer inWnt7aCre/+;Esr1f/f, but it was not significantly different from the lengthin Esr1f/f oviducts. These findings suggest that selectiveablation of Esr1 in the oviductal epithelial cells does notaffect epithelial cell proliferation but alters the length of thecilia in the oviduct.

A recent study showed that a global deletion of akinesin superfamily (Kif) gene caused an increase in ciliarylength, resulted in an ineffective clearance of the mucusand cell debris in the oviduct (34). Therefore, to assesswhether the embryo transport defect inWnt7aCre/+;Esr1f/f

animals is possibly caused by a deficit in ciliary function,wemeasuredCBF in ex vivo culturedoviducts at 0.5 dpc.Abox-and-whisker plot of the CBFs showed a significantdifference in the median values of the beat frequenciesbetween Esr1f/f and Wnt7aCre/+;Esr1f/f oviducts (Fig. 2J).In addition, the number of cells at each frequency waspresented in a histogram, which showed that there weremore ciliated cells inWnt7aCre/+;Esr1f/f that beat at lowerfrequencies, compared with those in Esr1f/f animals.Moreover, we evaluatedwhether an increase in the ciliarylength in Wnt7aCre/+;Esr1f/f oviducts was due to an aber-rant expression of certain kinesin family genes. Kif9 andKif19, but not Kif3a or Kif6, were expressed at a signifi-cantly lower level inWnt7aCre/+;Esr1f/f compared toEsr1f/f

oviducts (Fig. 2K). In addition, Foxj1 (ciliogenesis marker)level was also significantly less in Wnt7aCre/+;Esr1f/f

compared to Esr1f/f oviducts. These results suggest that alack of ESR1 in the oviductal epithelial cells results in anincrease in the ciliary length, which alters ciliary beatfunction; thus, such a conditionmay be an explanation forthe cause of defective embryo transport.

The WNT/CTNNB1 signaling pathway in theoviduct is regulated by E2

To identify downstream molecular mechanisms of E2 sig-nals regulating cilia function, we evaluated the potentialsignaling pathways that are shown to be involved in ciliafunction, one of which is the WNT/CTNNB1 pathway(35). WNT family members are secreted glycolipoproteinsthat signal through both canonical CTNNB1 and non-canonical pathways. CTNNB1, a transcriptional coac-tivator encoded by the Ctnnb1 gene, regulates embryonicdevelopment and adult homeostasis (36). Recent studies inzebrafish and mice demonstrated that WNT/CTNNB1signaling is crucial for ciliogenesis andairwaymulticiliatedcell differentiation (35). Thus, we sought to test whether atransport defect in the Wnt7aCre/+;Esr1f/f oviduct wasdue to altered WNT/CTNNB1 signaling in the oviduct.Microarray analysis of the oviducts collected fromEsr1f/f

and Wnt7aCre/+;Esr1f/f animals at 0.5 dpc showed that34% (21/61) of WNT/CTNNB1 signaling-related geneswere differentially expressed in the absence of epithelialESR1 (Table 1). In addition, we validated alteredexpression of transcripts that encompassed various

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WNT/CTNNB1 components using qRT-PCR analysis,including WNT ligands (Wnt7a and Wnt7b) and a WNTreceptor (Frizzled 10 or Fzd10), a transcription factor(Tcf3), and CTNNB1 direct target genes (Cdh1 andFgh18). All transcripts were significantly altered, exceptTcf3 and Ctnnb1, in the absence of epithelial ESR1 ex-pression (Fig. 3A). Although the overall transcript level ofCtnnb1was not different between Esr1f/f andWnt7aCre/+;Esr1f/f oviducts, theprotein expressionpatternofCTNNB1differed (Fig. 3B). IHC analysis showed that the localiza-tion of CTNNB1 was restricted only to the lateral (arrow-heads), not apical (arrows),membranes of epithelial cells inboth the ampulla and the isthmus in Esr1f/f animals. In

contrast, however, in the Wnt7aCre/+;Esr1f/f oviducts,CTNNB1 was also localized at the apical membrane ofthe epithelial cells. This result suggests that the tran-script is not affected, but the intracellular localization ofCTNNB1 protein is disrupted by the lack of ESR1 sig-naling in the oviductal epithelial cells.

To further assess the impact of the natural physiologicresponse to ovarian hormones on WNT/CTNNB1 sig-naling in the various regions of the oviduct, we collectedandanalyzedRNAfromthe3different regions of oviductsfrom wild-type animals at each stage of the estrous cycle[diestrus (lowest E2 level), proestrus (highest E2 level),metestrus, and estrus] by RT-PCR. It is known that E2

Figure 2. Loss of ESR1 in the oviductal epithelial cells leads to impaired embryo transport. A) IHC analysis of ESR1 expression inthe infundibulum, ampulla, and isthmus in control (Esr1f/f) and Wnt7aCre/+;Esr1f/f oviducts. B) Percentage of wild-type embryosretrieved from the oviduct and the uterus in Esr1f/f and Wnt7aCre/+;Esr1f/f females at 3.5 dpc (n = 5–6 recipients/genotype; totalviable embryos retrieved = 18 from Esr1f/f and 15 from Wnt7aCre/+;Esr1f/f recipients). Ovd; oviduct. C , D) Expression ofproliferative (Ki67) marker using IHC analysis (C) and quantification of Ki67-positive epithelial cells (D) in Esr1f/f andWnt7aCre/+;Esr1f/f

oviducts at 0.5 dpc (n = 4–5 mice/genotype, graphs represent means 6 SEM). Data represent 14,102 epithelial cells counted inEsr1f/f and 11,991 cells in Wnt7aCre/+;Esr1f/f. E , F) H&E staining (E) and expression of ciliary (Ac a-tubulin) marker by using IHCanalysis (F) in Esr1f/f and Wnt7aCre/+;Esr1f/f oviducts at 0.5 dpc. G, H) Immunoblot analysis (G) and quantification (H) of wholeoviducts, indicating the levels of ciliary proteins including Ac a-tubulin, ARL13B, and TUBB4 normalized to b-actin in Esr1f/f andWnt7aCre/+;Esr1f/f animals at 0.5 dpc (n = 3 mice/genotype). I) Ciliary length measured from the infundibulum and ampullaregions of the oviduct sections stained with Ac a-tubulin in Esr1f/f and Wnt7aCre/+;Esr1f/f oviducts at 0.5 dpc. Data represent means6 SEM from a total of 3100 cells in Esr1f/f and 2019 cells in Wnt7aCre/+;Esr1f/f oviducts (n = 4 mice/genotype). *P, 0.05 vs. Esr1f/f,by unpaired Student’s t test. J ) CBF was measured from each ciliated cell in ex vivo cultures of Esr1f /f and Wnt7aCre/+;Esr1f/f

oviducts collected at 0.5 dpc and presented as Hz in a box-and-whisker plot. Number of cells at each designated frequency wasalso represented in a histogram. Data from 168–469 epithelial cells were recorded from each animal. *P , 0.05 vs. Esr1f/f (n = 3mice/genotype), Mann-Whitney U test. K) Levels of Kif3a, Kif6, Kif9, Kif19, and Foxj1 transcripts in the Esr1f/f and Wnt7aCre/+;Esr1f/f

oviducts at 0.5 dpc (n = 4–5 mice/genotype). *P , 0.05 vs Esr1f/f, unpaired Student’s t test.

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increases the expression of WNTs and CTNNB1 but de-creases secreted frizzled-related protein (SFRP)-2 (a neg-ative regulator ofWNT ligands in the female reproductivetract) inmouseuteri (37), and this E2-induced expression isnecessary for uterine cell growth.However, little is knownregardingE2 regulationofCTNNB1 inoviducts, except fora single study of an avian model (38). Ctnnb1 transcriptwas highly expressed in the infundibulum (where ciliatedcells are most abundant), gradually decreased in the am-pulla, and was detected at minimal levels in the isthmus(Fig. 3C). However, Ctnnb1 expression did not appear todiffer between the different stages of the estrous cycle,indicating ovarian hormones do not preferentially regu-late oviductal Ctnnb1 in a stage specific manner. Thenegative regulator of CTNNB1 Chibby1 (Cby1) (39) alsoshowed an expression pattern similar to that of Ctnnb1.In contrast, the expression of Sfrp2 was stage specificand highly expressed in the ampulla and was signifi-cantly increased during proestrus and estrus whencompared to diestrus. Expression ofAxin2 and Cdh1, directCTNNB1 targets, were significantly increased in the am-pulla regionatproestruscompared todiestrus.Similarly, theexpression of Fzd9 and Wnt2 was significantly elevated inthe ampulla at proestrus.Wnt7awas highly expressed in theisthmusregioncompared to the infundibulumandampulla,and its expression did not change at any stage of the estrouscycle. Wnt7b levels were highest in the infundibulum andgradually decreased in the ampulla and isthmus. These re-sults indicate that the expression of some of the WNT/CTNNB1 signaling transcripts are regulated in a physio-logic-region–specific and estrous-cycle–selective manner.

CTNNB1 is exclusively expressed in theepithelial cells of human fallopian tubes

To determine the appropriateness of our experimentalmouse model to human studies and whether CTNNB1signaling may have a clinical impact on cilia function inwomenaswell asmice,wenext assessed the localizationofCTNNB1 protein in ciliated epithelial cells from humanfallopian tubes. We found that CTNNB1 was especiallyenriched in the lateralmembrane of ciliated epithelial cells,as in the mouse samples (Fig. 4), and was not detected instromal and muscle cells. Epithelial cells also had positivestaining for the cilia marker Ac a-tubulin. These observa-tions indicate that CTNNB1 may function similarly inmouse oviduct and human fallopian tube cilia, therebyhaving the potential of impacting human health and fer-tility if dysfunctional.

Inhibition of the WNT/CTNNB1 signalingpathway disrupts embryo transport ex vivo

Because we observed that lacking ESR1 in the oviductalepithelial cells causedanaberrant expressionof transcriptsthat impact WNT/CTNNB1 signals, as well as alteredlocalization of CTNNB1 protein, we hypothesized thatESR1-mediated E2 responses during embryo transport aremediated by WNT/CTNNB1 signaling. To directly eval-uate whether the CTNNB1 signaling pathway is requiredfor embryo transport, we employed an ex vivo oviductculture model to compare transport distances of embryosin the presence or absence of theWNT inhibitor IWP2 (40).Oviducts were collected at 0.5 dpc with zygotes inside(Fig. 5A). Oviducts were uncoiled, placed onto theTranswell insert in a 24-well plate, and incubated withDMSO (vehicle control) or 5 mM IWP2 for 3 consecutivedays. Subsequently, the distances that the embryos trav-eled were measured as a percentage of the distance be-tween theAIJ and theUTJ. In a DMSO-treated control, theembryoswere able to travel;23.56 3.4% of the oviductallength, whereas embryos in oviducts treated with IWP2traveled a significantly shorter distance (7.56 4.3% of theoviductal length) (Fig. 5B). To ensure that the decreasedtransport observed in the IWP2 treatment group is relatedto the specific effectof the treatment,we confirmed that thelevel of the CTNNB1 target gene Axin2 was significantlylower in the presence of IWP2 compared to the DMSO-treatedcontrol (Fig. 5C). Inaddition, the embryos retrievedafter 3 d of ex vivo oviduct culture were at similar de-velopmental stages in both treatment groups, suggestingthat the treatment did not result in toxicity (Fig. 5D). Thesefindings show that inhibition ofWNT/CTNNB1 signalingreduces the oviduct’s ability to transport embryos.

Selective ablation of CTNNB1 in ciliatedepithelial cells does not disrupt the embryotransport in vivo

Several transcripts in the canonical WNT/CTNNB1pathway are altered in the absence of epithelial ESR1(Table 1). Therefore, to determine whether the canonical

TABLE 1. ESR1 regulates expression of canonical and noncanonicalWNT signaling-related genes

Gene Fold change P

Extracellular factorDkk1 22.27 0.0031Dkk3 22.81 0.0006Fzd3 2.11 0.0002Fzd10 4.71 0.0001Wnt5b 1.54 0.0057Wnt7a 7.05 0.0028Wnt7b 22.01 0.0058Wnt11 1.76 0.0030Wnt16 21.73 0.0006

CTNNB1 regulatorPpp2r2c 21.88 0.0003

Transcription factorSox9 22.65 0.0083Tcf1 2.43 0.0032Tcf3 1.63 0.0026

CTNNB1 target geneCd44 22.66 0.0027Cdh1 21.55 0.0054Ephb2 1.82 0.0061Fgf18 2.99 0.0003Id2 1.86 0.0014Lgr5 3.40 0.0005Rhou 1.53 0.0017Ppard 7.26 8.4E207

Fold change, Wnt7aCre/+;Esr1f/f vs. Esr1f/f.

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WNT/CTNNB1 pathway within the ciliated cells isdownstream of E2 action and mediates embryo transportthrough the oviduct in vivo, we generated a conditionalknockout mouse model where Ctnnb1 was selectivelyablated in ciliated epithelial cells by crossing Foxj1Cre+ toCtnnb1f/f animals. As CTNNB1 is the central protein forcanonical WNT/CTNNB1 signal, ablation of CTNNB1would disrupt the whole signaling cascade. First, weconfirmed that CTNNB1 protein is deleted specifically inciliated epithelial cells of the oviduct. InCtnnb1f/f controls,CTNNB1 was detected in the oviduct epithelium in-cluding both secretory and ciliated cells in ampulla andisthmus, as well as the epithelial cells of the uterus (Fig.6A). CTNNB1 staining was mainly found in the lateralplasmamembraneof epithelial cells,which is similar to thepattern detected in human fallopian tubes (Fig. 4). In theFoxj1Cre+;Ctnnb1f/f oviduct, CTNNB1 was not detected inciliated epithelial cells but was retained in secretory epi-thelial cells (Fig. 6A). Note that CTNNB1 deletion mainlytook place in the ampulla of the oviduct, as the isthmusregion has few ciliated cells, as indicated by Ac a-tubulinIHC (Fig. 1A).

We next evaluated whether the deletion of Ctnnb1 inciliated epithelial cells impairs the transport of embryos byretrieving them from the oviduct and theuterus at 3.5 dpc.There was no significant difference in the number of em-bryos retrieved from the uterus and oviduct betweenFoxj1Cre+;Ctnnb1f/f and Ctnnb1f/f females (Fig. 6B). In

addition, the total numbers of embryos per damaswell asthe development of embryos were comparable betweenFoxj1Cre+;Ctnnb1f/f and Ctnnb1f/f females (Fig. 6C, D).Further investigation revealed that, overall, ciliated epi-thelial cells thatwerepositive forAca-tubulin stainingdidnot appear to be different in Foxj1Cre+;Ctnnb1f/f comparedto Ctnnb1f/f oviducts (Fig. 6E). Collectively, our findingssuggest that lackingCTNNB1 in the ciliated epithelial cellsof the oviduct does not disrupt the transport or the de-velopment of the embryos, indicating that CTNNB1 in thesecretory oviductal epithelial cells may induce the signalsallowing embryo transport, summarized as a workingmodel in Fig. 7.

DISCUSSION

In this study, we showed that ESR1 was expressed in allcell layers of the mouse oviduct, whereas ESR2 was spe-cifically found in cilia of multiciliated epithelial cells.However, a global loss of Esr2 had no impact on the em-bryo transport function of the oviduct. On the contrary,selectiveablationofEsr1 in theoviductal epithelial cells ledto a transport defect, in part because of an overall re-duction of CBF, possibly caused by an increase in ciliarylength. We also determined that expression of WNT/CTNNB1 signaling-related genes in the oviduct was reg-ulated by E2. Recent findings indicate that the expression

Figure 3. Expression of transcripts (A) and protein (B) involved in the WNT/CTNNB1 signaling pathway in Esr1f/f and Wnt7aCre/+;Esr1f/f oviduct at 0.5 dpc and at each stage of the estrous cycle (C) in the infundibulum, ampulla, and isthmus of wild-typeoviducts. Representative images (B) show CTNNB1 IHC staining in Esr1f/f and Wnt7aCre/+;Esr1f/f oviducts at 0.5 dpc. Arrows:difference between the expression of CTNNB1 at the apical membrane of epithelial cells in Esr1f/f and Wnt7aCre/+;Esr1f/f

oviducts; arrowheads: expression of CTNNB1 at the lateral membrane of the epithelial cells. Expression values determined byqRT-PCR (Wnt7a, Wnt7b, Fzd10, Tcf3, Cdh1, Fgf18, Ctnnb1, Cby1, Sfrp2, Axin2, Cdh1, Fz9, and Wnt2) were normalized to Rpl7(n = 4–5 mice/genotype). Graphs represent means6 SEM. #P, 0.05 vs. Esr1f/f, unpaired Student’s t test; *P, 0.05, **P, 0.01 vs.the expression value within the same region of the oviduct at diestrus (lowest circulating E2).

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of CTNNB1 is increased in the ampulla region of fallopiantubes of patients with diseases, such as salpingitis, andectopic pregnancy (41). Together with our findings, theseobservations suggest that aberrant expression of CTNNB1in the fallopian tubes could result in ectopic pregnancy. Inthe mouse, inhibition of WNT/CTNNB1 signaling dis-rupted the embryo transport in an ex vivo oviduct culture.However, conditional removal of CTNNB1 in oviductalciliated epithelial cells had no effect on the embryo trans-port through the oviduct. Thus, our findings demonstrateexpressionWNT/CTNNB1 in oviductal ciliated epithelialcells is not required for embryo transport but rather ex-pressionof epithelialESR1 in theoviduct is essential for theproper ciliary function of the oviduct.

It has been reported that ESR2, which is detected inoviductal cilia, modulates the expression of calbindin-D28K (20), a signalingmolecule critical for Ca2+-regulatedciliary beating. Our findings, however, showed that de-letion of Esr2 had no impact on the transport of embryos.This suggests that ESR2 signaling, and its downstreampathways, appear not to be involved in the regulation ofciliary beating of the oviduct during the transport ofembryos.

Previous studies have demonstrated that pharmaco-logical inhibitionofESR1 functionusinganESRantagonistsuppresses the proliferation of oviductal ciliated cells inneonatal mice (17). In addition, extensive loss of ciliatedcells and shortening of ciliary length are observed in rab-bits after ovariectomy (42), suggesting that loss of ovarianhormones dramatically affects ciliated cell morphologyand growth. Moreover, E2 induces expression of Foxj1(a central regulator of themotile ciliogenesis) in themouseoviduct and rhesus macaque fallopian tubes (17, 43). Ad-ditionally, the number of ciliated epithelial cells in the ef-ferent duct in male is also regulated by E2 signaling as aglobal deletion of Esr1 (Esr12/2) causes a decrease in thenumber of motile cilia compared to Esr1+/+ mice (44).These findings indicate that E2 regulates the turnover ofciliated epithelial cells through ESR1 in both male and

female reproductive tracts. Recently, a mouse modellackingkinesin superfamily (Kif192/2) showedthat ciliaofKif192/2oviduct aredoubled in length comparedwith thecontrol littermates (34). As KIF19 is a microtubule-depolymerizing kinesin expressed at the ciliary tips,without KIF19, the depolymerizing process is disrupted,leading to an increase in ciliary length (34). The increasedciliary length in Kif192/2 mice contributes to defectiveciliary beating, resulting in ineffective fluid flow in theoviduct. In our mouse model, we found that the absenceof epithelial ESR1 in the oviduct causes decreases inKif19, Kif9, and Foxj1 transcripts. Thus, it is possible thatblunted expression of Kif19 and Kif9 could cause the in-creased ciliary length and aberrant CBF we observed inWnt7aCre/+;Esr1f/f oviducts, leading to impaired embryotransport.

Although the ciliary length is increased in the in-fundibulum region of Wnt7aCre/+;Esr1f/f oviducts, thelevels of ciliary proteins, includingAc a-tubulin, ARL13B,and TUBB4, tended to be augmented but not significantlydifferent from those of Esr1f/f oviducts. This finding indi-cates that the observed 14% increase of ciliary length inthe infundibulum (2.61 mm in Esr1f/f vs. 2.98 mm inWnt7aCre/+;Esr1f/f oviduct) does not alter the levels ofoverall ciliary protein expression in the whole oviducts.

Figure 5. Inhibition of WNT/CTNNB1 signaling pathway leadsto an attenuation of the ex vivo embryo transport. A)Procedure for ex vivo oviduct culture in vehicle (DMSO) orIWP2 at a dose of 5 mM, for 3 consecutive days. B) The distancethe embryos traveled from AIJ to UTJ in the presence orabsence of IWP2. The distances were measured from AIJ toUTJ and calculated as a percentage of the distance, in whichthe embryos that travel to the UTJ are 100%. C) Axin2 (targetgene of the canonical WNT/CTNNB1 pathway) transcript inthe oviducts that were cultured in the presence or absence ofIWP2 for 3 consecutive days (n = 4–5 mice/group, graphsrepresent means SEM). *P , 0.05 vs. vehicle control, unpairedStudent’s t test. D) Representative images of embryos flushedfrom the oviducts that were treated with DMSO or IWP2 for 3consecutive days.

Figure 4. Representative images of IHC staining for CTNNB1and Ac a-tubulin in the epithelial cells of human fallopiantubes (n = 3 individuals).

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Several studies reported that exogenous treatmentwithE2 can increase or decrease the embryo transport ratedepending on the dose administered (45–47). In rodents,the effects of E2 on embryo transport in the oviduct havebeen reported to be mediated through both genomic andnongenomic actions (48–50). Collectively, this indicatesthat E2modulates theCBF in the oviduct. In this study,wefound that in the absence of ESR1 in the epithelial cells, themedian of CBFs was significantly lower when comparedto the controls. In addition, there were a number of ciliatedcells that beat at higher frequencies (.20 Hz), which couldpotentially alter the net transport rate of the embryos. Hy-drodynamic force (or the fluid flow) is one of the factorsaffecting the CBFs (51, 52). Several studies in rabbits andhumansdemonstrated thatE2 increases the fluid flow in theoviduct (53, 54).Moreover, our recent findings indicate thata lack of ESR1 in the epithelial cells leads to an alteration insecretory proteins produced by the oviduct, includingproteases andprotease inhibitors, resulting inanembryonicdeath within the oviduct (22). Taken together, these dataimply that the absence of ESR1 in the oviductal epithelialcells could potentially disrupt fluid secretion, whichwouldsubsequentlyalter the transportof the embryosasa result ofcumulative loss of fluid flow and irregular CBFs.

Upon binding of a WNT ligand to its receptor, FZD,CTNNB1 translocates into the nucleus and stimulatestarget gene expression through interactionswith the T-cellfactor/lymphocyte enhancer factor-1 transcription factor(55). E2 increases expression of WNT4, WNT5A, andCTNNB1 inmouse uterine epithelial cells (38) and inducesthe nuclear translocation of CTNNB1 and formation of the

CTNNB1/LEF1 complex in rat uterine and MCF-7 cells(56, 57). Inversely, uterine expression of the negative reg-ulator of WNT/CTNNB1 signaling SFRP2 is suppressedby E2 treatment (37). In this present study, we found thatexpression of many WNT/CTNNB1 signaling moleculeswere significantly altered after deletion of ESR1 in ovi-ductal epithelial cells. In addition, CTNNB1 protein wasdetected on the apical membrane of epithelial cells in theabsence of epithelial ESR1, whereas the apical localizationof CTNNB1 was minimally detected in the control an-imals (either Esr1f/f or Ctnnb1f/f).

WNT/CTNNB1 signaling has been linked to ciliafunction (58). Mice with loss of Cby, a repressor of canon-ical WNT/CTNNB1-dependent gene transcription, de-velop sinusitis and otitis because of amarked reduction inthenumberofmotile cilia, leading todefectivemucociliaryclearance (39, 59).Cby2/2males and females are subfertile(39). These results suggest thatdysregulationof theWNT/CTNNB1 pathway can result in defective motile cilia. Be-cause global loss of CTNNB1 (Ctnnb12/2) is embryoniclethal due to a gastrulation defect (60), this mouse modelcannot be used to study CTNNB1 function in the oviduct.Similarly, mice carrying a tissue-specific deletion ofCTNNB1 in the stromal and muscle cell layers of femalereproductive tract (using Amhr2Cre/+;Ctnnb1f/f animals)cannot be used for such studies because of oviduct de-velopmental defects, including partial oviduct formation,failure of coiling, and lack of mucosal folding of the epi-thelial layer (61, 62).Wegenerated a conditional-knockoutmouse line lacking Ctnnb1 selectively in ciliated epithelialcells to test whether CTNNB1 is functionally required for

Figure 6. Loss of CTNNB1 in ciliated epithelial cells does not affect the transport of embryos within the oviduct. A) IHC analysisof CTNNB1 in the ampulla, isthmus, and uterus of control (Ctnnb1f/f) and Foxj1Cre+;Ctnnb1f/f females. Note that the expression ofCTNNB1 was not detected in the ciliated epithelial cells of Foxj1Cre+;Ctnnb1f/f females. B) Percentage of the embryos (bothmorulae and blastocysts) retrieved from the oviduct and the uterus in Ctnnb1f/f and Foxj1Cre+;Ctnnb1f/f females at 3.5 dpc. C) Totalnumber of embryos in Ctnnb1f/f and Foxj1Cre+;Ctnnb1f/f females at 3.5 dpc. Graphs represent means 6 SEM (n = 6–9 mice/genotype). D) Representative images of embryos flushed from the oviducts and uterus of Ctnnb1f/f and Foxj1Cre+;Ctnnb1f/f femalesat 3.5 dpc. E) Ciliated epithelial cells of Ctnnb1f/f and Foxj1Cre+;Ctnnb1f/f oviducts indicated by Ac a-tubulin IHC.

1604 Vol. 31 April 2017 LI ET AL.The FASEB Journal x www.fasebj.org Vol.31,  No.4 , pp:1595-1607, March, 2017The FASEB Journal. 134.121.125.9 to IP www.fasebj.orgDownloaded from

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the function of ciliated cells during embryo transport. Toour surprise, there was no embryo transport defect in theabsence of Ctnnb1 in ciliated cells, which suggested thatCTNNB1 intrinsic to ciliated epithelial cells is not requiredduring embryo transport. However, CTNNB1 is still pre-sent in nonciliated cells and may affect ciliated cells viaparacrine signaling. This interpretation is supported byour ex vivo culture results clearly showing that inhibitionofWNT/CTNNB1 signaling disrupted the embryo trans-port. It is likely that the transport defect observed in the exvivo model resulted from the inhibitory effects on bothsecretory and ciliated epithelial cell functions. It was pre-viously reported that primary cilia are present in thenonciliated epithelial cells of human fallopian tubes (63)and WNT/CTNNB1 signaling is crucial for primary ciliafunction (64). Therefore, the function of nonciliated (se-cretory) epithelial cells could also be affected as IWP2 alsoinhibitsnoncanonicalWNTsignaling.Thus, thepossibilitythat the noncanonical WNT pathway may be involved inthe regulation of cilia and nonmotile function in the ex vivooviduct culture has to be considered.

A growing number of case reports suggest that theprolonged use of clomiphene citrate (Clomid; SanofiAventis, Gentilly, France) is linked with bilateral ectopicpregnancy (65–67). Clomid has been widely used morethanhalf a centuryby fertility clinics to induceovulation ininfertile women (65). It is a selective estrogen receptormodulator (SERM), and acts by binding to the ESRs andblocking E2 action (68), increasing the release of folliclestimulating hormone and luteinizing hormone (69). Thecause of bilateral ectopic pregnancy from the use of Clo-mid has not been investigated, but we can speculatewhether it is the result of disrupted E2/ESR1 signaling inthe fallopian tubes. Findings in rats suggest that animalstreated with Clomid showed delayed oocyte transportwithin the oviduct (70). In this study, we showed thatdisruption of E2 signal in the epithelial cells of the mouseoviduct causes an embryo transport defect. Together,these findings indicate that aberrant E2 signaling, either via

useofESRantagonists orviadisruptionofESR1 itself in theepithelial cells of the oviduct impairs embryo transport,increasing the risk of ectopic pregnancy.

In summary, our study provides evidence that E2signal via ESR1 is clearly required for the embryotransport function of the oviduct. The details of E2-regulated molecular mechanisms in the multiciliatedepithelial cells and secretory cells of the oviduct willrequire further analysis.

ACKNOWLEDGMENTS

The authors thank Sylvia Hewitt and Karina Rodriguez [U.S.National Institutes of Health, National Institute of EnvironmentalHealth Sciences (NIEHS)] for critical reading of this manuscript,Carmen Williams (NIEHS) for constructive advice for theproject, Page Myers (NIEHS) for the oviduct transfer procedure,Jeff Tucker (NIEHS) for technical assistance, NIEHS HistologyCore for ESR2 IHC staining, and Research Histology CoreFacility (Department of Pathology, Stony Brook UniversitySchool of Medicine) for the human fallopian tube samples. Thiswork is supported by the start-up fund from College of VeterinaryMedicine (Washington State University; to W.W.) and Intramuralresearch Grant 1ZIAES70065 from NIEHS (to K.S.K.). Theauthors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

W.Winuthayanondesigned theresearch;W.Winuthayanon,S. Li, and S. R. S. O’Neill performed research and analyzeddata; S. Li and W. Winuthayanon wrote the paper; and K.-I.Takemaru, Y. Zhang, M. J. Holtzman, and K. S. Korachcontributed newprotocols, samples, andmouse lines crucialfor the study.

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Figure 7.Working model illustrates E2 and CTNNB1 signals in the oviductal ciliated (blue) and secretory (orange) epithelial cellsregulating embryo transport. A) In the presence of ESR1, WNT/CTNNB1 is normally expressed, leads to a normal embryotransport, regardless of ESR2 expression in the ciliated epithelial cells. B) In the absence of ESR1, expression of WNT/CTNNB1,Kif9, Foxj1 are aberrantly expressed, ciliary length and beating are altered, causing impaired embryo transport. C) In thepresence of ESR1, ex vivo attenuation of WNT/CTNNB1 signals in oviductal epithelial cells disrupts embryo transport, indicatingthat the WNT/CTNNB1 pathway is a potential downstream mediator of E2-regulation of embryo transport through the oviduct.D) Loss of CTNNB1 specifically in the ciliated epithelial cells does not affect oviductal embryo transport, indicating that E2modulates WNT/CTNNB1 signals in secretory epithelial cells, and also acts through the noncanonical WNT signaling pathway orother downstream pathways in the ciliated epithelial cells, to control the embryo transport.

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Received for publication October 7, 2016.Accepted for publication December 19, 2016.

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