BIOLOGY OF REPRODUCTION (2012) 86(4):120, 1–10Published online before print 1 February 2012.DOI 10.1095/biolreprod.111.096339

Ciliary Activity in the Oviduct of Cycling, Pregnant, and Muscarinic ReceptorKnockout Mice1

Katharina Noreikat,3,4 Miriam Wolff,5 Wolfgang Kummer,5 and Sabine Kolle2,3,6

3Institute of Veterinary Anatomy, Histology, and Embryology, Justus-Liebig University, Giessen, Germany4Department of Anesthesiology and Intensive Care Medicine, University of Leipzig, Leipzig, Germany5Institute of Anatomy and Cell Biology, Justus-Liebig University, Giessen, Germany6Department of Urology, University of Munich, Munich, Germany

ABSTRACT

The transport of the oocyte and the embryo in the oviduct ismanaged by ciliary beating and muscular contractions. Becausenonneuronally produced acetylcholine influences ciliary beatingin the trachea via the muscarinic receptors M2 and M3, wesupposed that components of the cholinergic system may alsomodulate ciliary activity in the oviduct. To address this issue, weanalyzed the expression profile of muscarinic receptors(CHRMs) in the murine oviduct by RT-PCR and assessed ciliarybeat frequency (CBF) and cilia-driven particle transport speed(PTS) on the mucosal surface of opened oviductal segments incorrelation with histomorphological investigations. RT-PCR oflaser-assisted microdissected epithelium revealed expression ofChrm subtypes Chrm1 and Chrm3. In opened isthmic segments,particle transport was barely seen, correlating with a signifi-cantly lower number of ciliated cells compared to the ampulla.In the ampulla, basal PTS and CBF were high (71 lm/sec and 21Hz, respectively) both in cycling and pregnant wild-type miceand in mice with targeted deletion of the Chrm genes Chrm1,Chrm3, Chrm4, and Chrm5. In contrast to the trachea, wherebasal ciliary activity was low and largely enhanced bymuscarinic stimulation, muscarinic agonists and antagonistsdid not affect the high ampullar PTS. Our results imply that thishigh oviductal autonomous ciliary activity is independent fromthe intrinsic cholinergic system and serves to maintain optimalclearance of the tube throughout all stages of the estrous cycleand early pregnancy.

ciliary transport, nonneuronal cholinergic system, oviduct,pregnancy

INTRODUCTION

Transit through the oviduct is driven by two majormechanisms: smooth muscle contraction and ciliary activity.Although the relative importance of these mechanisms is stillnot fully clear, several lines of evidence indicate that ciliaryaction may play a dominant role in the transport of the gametesand the embryo [1]. Supportive of this notion, pharmacologicalinhibition of muscular activity has no impact upon total transittime through the ampulla [2, 3], and women with immotile cilia

syndrome suffer from subfertility [4]. Still, ciliary activity isnot absolutely mandatory for successful embryo transport,because successful pregnancies have also been reported inimmotile cilia syndrome and when an excised ampullarsegment is reimplanted in a reverse direction [4, 5].

In addition to its role in gamete and embryo transport, thefluid stream directed toward the uterus generated by the ciliarybeat may serve a clearance function analogous to themucociliary clearance of the ciliated airway epithelium, whereairway-lining fluid and mucus, together with inhaled particles,are transported toward the larynx for expulsion. Consequently,regulation of oviductal ciliary transport is expected to have afar-reaching impact upon fertility as well as structural andfunctional integrity of the uterine tube. Still, knowledge islimited regarding the mechanisms governing ciliary transport inthe oviduct, with the majority of experiments performed oncultured cells [6]. We recently developed a method to measurecilia-driven particle transport and ciliary beat frequency (CBF)in freshly explanted murine airways [7, 8] and bovine oviducts[9], where ciliated cells are still embedded in their physiolog-ical surroundings and collectively generate a directed fluidstream. In the present study, we extend this model to themurine oviduct to enable the use of transgenic animals forelucidating the role of specified gene products in regulation ofoviductal ciliary function.

In the murine trachea, ATP, 5-hydroxytryptamine (5-HT;serotonin), and acetylcholine (ACh) acutely stimulate bothparticle transport speed (PTS) and CBF, and deficiency ofcertain muscarinic ACh receptor subtypes results in reducedbasal PTS and CBF, indicating a role of endogenous ACh inmaintenance of basal ciliary function [7, 8]. Although not fullyidentical, oviductal and airway ciliated cells share manystructural, functional, and molecular features [10]. Theseinclude ATP-triggered increase in intracellular free Ca2þ

concentration via activation of P2Y receptors (purinergicreceptors, G-protein-coupled receptors stimulated by nucleo-tides such as ATP, ADP, UTP, and UDP) [11, 12] and localproduction of ACh by ciliated cells [13, 14]. Hence, we set outto determine which muscarinic ACh receptors (CHRMs) areexpressed in the mouse oviduct, what impact their genedeficiency has upon ciliary transport, and to what extent ATP,5-HT, and ACh acutely influence ciliary function. Thefunctional studies were correlated with structural analyses ofampullar and isthmic segments in the various stages of theestrous cycle and during pregnancy.

MATERIALS AND METHODS

Animals

For the experiments, female specified-pathogen-free C57BL/6 wild-type(WT) mice and mice with gene deficiency for muscarinic receptor (Chrm)

1Supported by the ‘‘Deutsche Forschungsgemeinschaft’’ (DFG KO1398/5-1).2Correspondence: Sabine Kolle, Department of Urology, University ofMunich, Marchionistr. 15, 81377 Munich, Germany.E-mail: sabine.koelle@med.uni-muenchen.de

Received: 16 September 2011.First decision: 28 October 2011.Accepted: 11 January 2012.� 2012 by the Society for the Study of Reproduction, Inc.eISSN: 1529-7268 http://www.biolreprod.orgISSN: 0006-3363

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subtypes Chrm1, Chrm3, Chrm4, and Chrm5 (Chrm1�/�, Chrm3�/�, Chrm4�/�,and Chrm5�/�, respectively; age, .10 wk) were used. The generation ofChrm1�/�, Chrm3�/�, Chrm4�/�, and Chrm5�/� mice has been describedpreviously [15–18]. The mice were obtained from Dr. Jurgen Wess (NationalInstitute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Formorphological analyses of the oviduct and functional analyses of the trachea,only WT mice (oviduct, females .10 wk; trachea, females and males .15 wk)were investigated. All mice were held under specified-pathogen-free conditionsbefore experiments. All experiments were performed in accordance with theGerman animal protection law and in accordance with the Guide for the Care andUse of Laboratory Animals. In total, 107 C57BL/6, 5 Chrm1�/�, 4 Chrm3�/�, 4Chrm4�/�, and 5 Chrm5�/� mice were investigated.

Mating of WT Mice

Occurrence of mating was determined by the presence of a vaginal plug.Mice were examined twice a day for the presence of vaginal plugs (indicationfor copulation). Plug controls (indication for copulation) took place twice a day.Pregnancy was proved by the documentation of 1-, 2-, or 4-cell embryos in thedissected, but closed, oviduct using an Olympus BX61WI light microscopeequipped with a 203 water-immersion objective and a monochrome CMOScamera (EHD Imaging GmbH).

Determination of Cyclic Stage

Cyclic stages were determined by analyses of vaginal smears, which weretaken after death in opened vaginas by a Q-tip (Velvera). After drying, vaginalsmears were fixed in methanol for 10 min (Roth) and stained with Giemsasolution (Merck) for 30–40 min. Each cyclic stage is characterized by thepresence of specific cells. Thus, during proestrus, parabasal cells and surfacecells predominate, whereas an accumulation of flat surface cells without nucleiis seen during estrus. Leukocytes are the main cell population during metestrus.In the course of diestrus, cell numbers distinctly decrease, and the vaginalsmears mainly consist of mucus.

Light Microscopy

Oviducts of 18 female mice were obtained immediately after death.Ampulla and isthmus of three mice each in proestrus, estrus, metestrus, anddiestrus as well as Day 1 and Day 2 of pregnancy were examined. Day 1 andDay 2 of pregnancy were chosen, because the mouse embryo stays in theoviduct for 2 days and then enters the uterus. For hematoxylin and eosin (H&E)staining and immunohistochemistry, specimens were fixed in Bouin fluid for 6–8 h, dehydrated in a graded series of ethanols, and embedded in paraffin.Sections (thickness, 4 lm) were cut on a Leica microtome. For H&E stainingand immunohistochemistry, specimens were stained with H&E and investigat-ed with a Leica DSM 2500 microscope.

Determination of Relative Number of Ciliated Cells inAmpulla and Isthmus

Cell counting of ciliated and secretory cells was performed both in theampulla and in the isthmus of WT mice using sections stained with H&E. Intotal, oviducts of 24 animals were investigated. Ampullae and isthmus of fourdifferent animals of each cyclic stage, four animals at Day 1 of pregnancy, andfour animals at Day 2 of pregnancy were counted. The slides were analyzedusing a Leica DM 1000 light microscope equipped with a 1003/1.25 oil RCimmersion objective (Olympus). Only cells that could be unequivocallyallocated to a specific cell type were taken into account.

Immunohistochemistry

For the localization of ciliated cells in the ampulla and isthmus of themurine oviduct during the estrous cycle and early pregnancy, the immunohis-tochemical localization of b-tubulin was additionally used. Sections (thickness,4 lm) of ampulla and isthmus were deparaffinized in xylene and rehydrated ina descending sequence of ethanols (100%–50% [vol/vol]) and distilled water.After that, sections were washed in PBS. Unspecific protein-binding sites weresaturated by 1-h incubation with 10% horse serum (Perbio Science), 0.5% (vol/vol) polyoxyethylene sorbitan monolaurate (Sigma Chemical Co.), and 0.1%(wt/vol) bovine serum albumin (Sigma-Aldrich) in 0.02 M PBS (pH 7.4)containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4�2H2O, and 2 mMKH2PO4. Then, the sections were incubated for 1 h at room temperature withthe mouse monoclonal b-tubulin IV antibody (1:250, clone ONS1A6;BioGenex), which was directly labelled with the Alexa Fluor Labeling Kit555 (Invitrogen) according to the manufacturer’s protocol (1 lg of

immunoglobulin G with 10 ll of component A/B). Negative controls weredone by using the nonrelevant monoclonal anti-mouse antibody directedagainst villin (1:75, clone ID 2C3; Beckman Coulter). Sections were rinsed,postfixed in buffered 4% paraformaldehyde (Merck) for 10 min, rinsed again inPBS, and coverslipped with carbonate-buffered glycerol (pH 8.6). Evaluationwas done with an epifluorescence microscope (Zeiss) equipped with a 545- to610-nm barrier filter.

Scanning-Electron Microscopy

The three-dimensional morphology and localization of ciliated andsecretory cells in the ampulla and isthmus during the estrous cycle andpregnancy were investigated by scanning-electron microscopy. For thispurpose, oviducts of 15 mice were freed from the surrounding mesosalpinximmediately after death and were opened using scissors for iridectomy (FineScience Tools). In the following, specimens were put on a cork plate and fixedwith insect needles. Oviducts were then fixed in 1% glutaraldehyde (vol/vol) inSorensen buffer (pH 7.4, 1:5 solution of 0.07 M KH2PO4 and 0.07 MNa2HPO4�2H2O) at 48C for 24 h. After two washes in Sorensen buffer,specimens were dehydrated in an ascending series of acetone (10%, 20%, 30%,40%, 50%, and 60% twice for 5 min each; then 70%, 80%, and 90% for 1 heach; and then 100% [all vol/vol] for 12 h). In the following, the oviducts weredried in a Union Point Dryer CPD 030 (Bal-Tec) by using liquid CO2 astransitional fluid. After being dried, specimens were coated with 12 nm of goldpalladium by a Union SCD 040 sputtering device (Bal-Tec). Scanning-electronmicroscopy was performed with the Zeiss DSM 950 scanning-electronmicroscope at magnifications from 2003 to 10 0003.

Measurement of PTS

For measuring the PTS, the proximal segment of the ampulla (2 mm) and amidsegment of the isthmus (3 mm) were transferred to a Delta T Culture Dish(Bioptechs) in which the glass bottom was covered with a thin layer of Sylgardpolymer (Dow Corning) and that was filled with 2 ml of cold Hepes buffer.Specimens were longitudinally opened and then gently rinsed with cold Hepesbuffer followed by exchange with 1.5 ml of fresh warm buffer, submerging theoviduct. The culture dish was transferred to the Delta T stage holder 45 minafter the animal’s death and was held constant at 378C. Before each experiment,normal ciliary beating was assessed with an UMPLFL1003W/0.70 water-immersion objective (Olympus).

For measuring of PTS, 3.0 ll of Dynabeads (9 3 10�6 polysterene beads)with a diameter of 2.8 lm (Invitrogen Dynal AS) were added to the Hepesbuffer. To avoid settling of particles, the suspension was mixed with the bufferat least 1 min before measurement, which started 55 min (ampulla) after death.In the ampulla, the basal PTS was measured eight times every 3 min, followedby a minute-by-minute interval after addition of ATP (10�4 M; purinergicreceptor agonist), 5-HT (10�4 and 10�6 M; muscarinic receptor agonist),atropine (10�6 M; muscarinic receptor antagonist), muscarine (10�4 M;muscarinic receptor agonist), or ACh (10�6 M, muscarinic and nicotinicreceptor agonist; all from Sigma-Aldrich). In the isthmus, particle transport waslacking; therefore, measurements of PTS were recorded only four times every 3min without adding any agents. Images were taken with an Olympus BX61WIlight microscope equipped with a 203 water-immersion objective and amonochrome CMOS camera (EHD Imaging GmbH) using the analysissoftware StreamPix (Norpix, Inc.). At each time point, 200 images (640 3

512 pixels, 12 bit) were taken at a frame rate of 49.32 images/sec. The originalfilms were converted from 12- to 8-bit gray scale and were used to track theDynabeads by an automatic tracking procedure. Only tracks that could befollowed over a length of at least 10 frames and did not deviate more than 15%from the direct connection between start point and end point were included infurther calculations.

The basal PTS was measured in the oviduct of cycling mice (total, n¼ 30;proestrus, n ¼ 7, estrus, n ¼ 14; metestrus, n ¼ 5, and diestrus, n ¼ 4), earlypregnant mice (total, n ¼ 10; Day 1 of pregnancy, n ¼ 5; and Day 2 ofpregnancy, n¼ 5), and in muscarinic receptor (Chrm) knockout mice (total, n¼21; Chrm1�/�, n¼ 5; Chrm3�/�, n¼ 8; Chrm4�/�; n¼ 4; and Chrm5�/�, n¼ 4).

Additionally, the effects of agonists and antagonists of muscarinic receptorswere investigated. Thus, the effects of ATP (10�4 M) and atropine (10�6 M), anantagonist of muscarinic receptor activation, were investigated in cycling mice(n¼ 8), mice on Day 1 of pregnancy (n¼ 4), and muscarinic receptor (Chrm)knockout mice (Chrm1�/�, n ¼ 5; Chrm3�/�, n ¼ 4; Chrm4�/�, n ¼ 4; andChrm5�/�, n¼4). As agonists of the muscarinic receptors, muscarine (10�4 M),ACh (10�6 M), and 5-HT (10�4 and 10�6 M) were used. The numbers ofcycling animals investigated were as following: muscarine, n¼ 4; ACh, n¼ 6;and 5-HT, n¼ 8 (with 4 at each concentration). The utilized concentrations ofagonists and antagonists were previously found to exert maximal effects on

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ciliated cells of another murine epithelial surface (i.e., the tracheal epithelium)[7, 8].

For comparing the ciliary activity in the oviduct and in the trachea,additional experiments using the murine trachea (n ¼ 6) were performed. Theexperimental setup for the measurement of PTS in the trachea was done asdescribed above with the exception of four modifications due to the anatomyand physiology (i.e., lower ciliary activity) of the trachea: 1) Images were takenbetween two cartilages to prevent large differences in image brightness, 2) thetemperature was kept at 328C instead of 378C, 3) the amount of Dynabeads wasreduced to 6.9 3 10�6, and 4) the interval between the measurements fordetermining the basal PTS was increased to 5 min.

Measurement of CBF

The general experimental setup for determining CBF was similar to thatdescribed for the measurement of the PTS except that images were taken in theabsence of Dynabeads using a 403 water-immersion objective (Olympus). Intotal, 1000 images (640 3 480 pixels) were taken at a frame rate of 105 images/sec. Ten ciliated cells per field of view were designated as ‘‘area of interest’’ foreach time series. Differences in brightness, produced by ciliary beating, wererecorded using the software Image-Pro Plus (MediaCybernetics, Inc.). CBF wasthen determined by Fourier transformation using AutoSignal software (SystatSoftware GmbH).

Basal CBF was determined in the oviductal ampulla (n ¼ 8) and in thetrachea (n¼ 6). The influence of ATP (10�4 M) and atropine (10�6 M) on CBFwas investigated in the oviductal ampulla and in the trachea in eight and sixanimals, respectively.

RT-PCR of Oviductal Epithelium after Laser-AssistedMicrodissection

We performed RT-PCR on oviductal segments (ampulla, isthmus, anduterotubal junction) and on microdissected epithelium. The procedure isdescribed here for microdissected samples, and modifications of the techniqueapplied to oviductal segments are given in parentheses.

Cryosections (thickness, 10 lm) from oviducts obtained from five micewere mounted on polyethylene naphthalate membrane-covered glass slides.After hemalaun staining for 45 sec, the sections were rinsed for 2 min in tapwater and subsequently immersed in 70%, 96%, and 100% ethanol.Microdissection of oviductal epithelium was performed using the PALMMicroBeam System (Carl Zeiss). Laser-dissected tissues were catapulteddirectly into the cap of a 0.5-ml microfuge tube by a laser pulse. Lysis buffer(Buffer RLT; Qiagen) was added, and the samples were snap-frozen in liquidnitrogen. After adding 5 ll of carrier RNA (4 ng/ll), total RNA was extractedwith the RNeasy Micro Kit (Qiagen) (for oviductal segments, carrier RNA wasnot used) according to the manufacturer’s protocol. Contaminations withgenomic DNA were removed by digestion with desoxyribonuclease I (RNase-Free DNase Set; Qiagen) for 5 min at room temperature (for oviductalsegments, deoxyribonuclease I [Invitrogen] was used for 25 min at 378C). RNAwas reverse transcribed using murine leukemia virus reverse transcriptase(Applied Biosystems) for 75 min at 438C (for oviductal segments, superscript IIreverse transcriptase [Invitrogen] was used for 50 min at 428C). The cDNAswere amplified with gene-specific primer pairs for Chrm subtypes 1–3 (primer

pairs, Chrm1, Chrm2, and Chrm3 [for oviductal segments, Chrm1–5, withprimer pairs Chrm1i, Chrm2, Chrm3i, Chrm4, and Chrm5]; Eurofins MWGOperon) (Table 1). For subsequent PCR, 4 ll of cDNA, 2.5 ll of buffer II, 2 llof MgCl2 (25 mmol/L), 0.5 ll of dNTP (10 mmol/L), 0.25 ll of each primer(20 pmol/L), and 0.2 ll of AmpliTaq Gold polymerase with 5 U/ll of GeneAmp (Applied Biosystems) (for oviductal segments, 0.1 ll) were supplementedwith H2O to a final volume of 25 ll. Cycling conditions were 10 min at 958C;50 cycles (for oviductal segments, 40 cycles) of 30 sec at 958C, 20 sec at 598C–608C, and 30 sec at 738C; and a final extension at 738C for 7 min. The PCRproducts were separated by electrophoresis on a 2% Tris-acetate-ethyl-enediaminetetra-acetic acid gel. The following controls were performed:Samples processed without reverse transcription were run to control forcontamination with genomic DNA for every primer pair, and primers for b2-microglobulin (B2m) were used as positive control for efficiency of RNAisolation and cDNA synthesis (Table 1). Sequencing of the PCR products wasdone by Eurofins MWG Operon.

Statistical Analysis

Multiple groups were compared with the nonparametric Kruskal-Wallistest. If the resulting P-value was less than 0.05, pairs of groups were comparedby Mann-Whitney test. The Wilcoxon test was used for comparison ofassociated samples if a preceding Friedman test revealed P , 0.05. Differenceswere rated significant if P � 0.05. Statistical analysis was performed using thesoftware SPSS 15.0 (SPSS, Inc.).

RESULTS

Distribution of Ciliated Cells and Secretory Cells in theMurine Oviduct

The murine ampulla was characterized by a thin smoothmuscle layer, a thin layer of subepithelial connective tissue,and long longitudinal mucosal folds displaying a highpercentage of ciliated cells in the epithelium (Fig. 1, A–F).Contrary to the ampulla, the isthmus showed a thick smoothmuscle layer, a very distinct layer of subepithelial connectivetissue, and circular mucosal folds with mainly secretory cells—single ciliated cells were only seen in the depth between thefolds (Fig. 1, G–L). During estrus, the number of secretorycells in the ampulla increased, and the secretory cells started toarise over the level of the ciliated cells (Fig. 1B). The activityof secretory cells in the ampulla as seen by cell protrusionsincreased from metestrus (Fig. 1C) and diestrus (Fig. 1D) to thefirst day of pregnancy (Fig. 1E). On the second day ofpregnancy, many emptied secretory cells were visible in theampulla (Fig. 1F, arrowhead). Contrary to the ampulla, nochanges were observed in the isthmic epithelial morphologyduring the cycle (Fig. 1, G–J). In the isthmus, a continuoushigh secretory activity was found during estrus (seen as

TABLE 1. Primer pairs used for RT-PCR.

Gene GenBank accession no. Primer Primer sequence (50!30) Amplicon length (bp) Annealing temp. (8C)

Chrm1 NM_007698 Forward cag tcc caa cat cac cgt ctt 441 60Reverse gag aac gaa gga aac caa cca c

Chrm2 NM_203491 Forward tgt ctc cca gtc tag tgc aag g 368 60Reverse cat tct gac ctg acg atc caa c

Chrm3 NM_033269 Forward gta caa cct cgc ctt tgt ttc c 244 60Reverse gac aag gat gtt gcc gat gat g

Chrm4 NM_007699 Forward cat cct ggt gat gct gtc ca 110 60Reverse tgc ctg agc tgg act cat tg

Chrm5 NM_205783 Forward cag cgt ccc atg agg atg ta 216 60Reverse cca tgg act gtg gga agt ca

Chrm1i T1 NM_001112697 Forward gag aag cac tga agc tga gga c 324 58T2 NM_007698 Reverse gag aag cac tga agc tga gga c 237

Chrm3i NM_033269 Forward cgt tct gac caa gtg acc aag c 186 60Reverse acc cag gaa gag ctg atg ttg g

B2m NM_009735 Forward atg gga agc cga aca tac tg 176 60Reverse cag tct cag tgg ggg tga at

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accumulation of fluid in the lumen) (Fig. 1H, arrowhead),during metestrus, and during pregnancy.

In contrast to ciliated cells, microvilli-bearing secretory cellslacked immunoreactivity to b-tubulin (Fig. 2, A and B). Bothin the ampulla and in the isthmus, the secretory cells werecharacterized by the appearance of microvilli, which weredistributed over the whole cell surface (Fig. 2, C and D). Thenegative controls performed by using the nonrelevant antibodyagainst villin regularly showed no signal (Supplemental Fig.S1, all Supplemental Data are available online at www.biolreprod.org). Independent of estrous cycle stage or an earlypregnancy, ciliated cells in the ampulla were significantly morefrequent (53%–81%) as compared to the isthmic region (1%–31%, P , 0.05) (Fig. 3). A significantly higher relativefrequency of ciliated cells was found at estrus (72%) incomparison to Day 1 (62%) and Day 2 (62%) of pregnancy (P, 0.05). In the isthmus, a significant difference occurred in thefrequency of ciliated cells between estrus (24%) and the othercyclic stages (proestrus, 6%; metestrus, 10%; and diestrus,10%) and the first day of pregnancy (8%, P , 0.05) (Fig. 3).

PTS in WT and Chrm-deficient mice

The basal PTS in the oviductal ampulla of WT mice was 656 4 lm/s (mean 6 SEM) (Figs. 4A and 5A and SupplementalMovie S1). In contrast, nearly no effective particle transportwas observed in the isthmus (Figs. 4B and 5B and

Supplemental Movie S2). Consequently, the following mea-surements were performed only at the ampulla. No differenceswere found in the basal PTS between cyclic stages or betweenWT and Chrm-deficient mice (Fig. 6). However, ATP (10�4

M) significantly (P , 0.05) decreased PTS, with a maximumafter 2 min of exposure time in WT (71 6 7 lm/sec vs. 66 6 8lm/sec) (Fig. 7) and Chrm3�/� (58 6 6 lm/sec vs. 49 6 3 lm/sec) (Fig. 7) mice. Chrm1�/�, Chrm4�/�, and Chrm5�/� miceshowed a tendency toward decreased PTS after exposure ofATP (Fig. 7). In WT mice, no alterations of PTS were evokedby atropine (10�6 M) (Fig. 7), muscarine (10�4 M) (Fig. 8A),ACh (10�6 M), and 5-HT (10�4 and 10�6 M) (Fig. 8B).Similarly, Chrm-deficient mice also showed no alteration inbasal PTS in response to atropine (Fig. 7). In contrast to thegeneral lack of effect on PTS and CBF, we noted contraction ofthe smooth muscles after administration of ATP, ACh, andmuscarine.

PTS in Pregnant WT Mice

The PTS of pregnant WT mice on Day 1 and Day 2 ofpregnancy did not significantly differ from that of nonpregnantmice (Fig. 6B). The effects of ATP (10�4 M) and atropine(10�6 M) on PTS in the ampulla were investigated only at Day1 of pregnancy (Fig. 7). The reason was the presence of verystrong muscular contractions on Day 2, which led to apronounced displacement of the mucosal surface, thereby

FIG. 1. H&E staining of ampulla (proestrus [A], estrus [B], metestrus [C], diestrus [D], Day 1 of pregnancy [E], and Day 2 of pregnancy [F]) and isthmus(proestrus [G], estrus [H], metestrus [I], diestrus [J], Day 1 of pregnancy showing a zygote with a mitotic spindle [arrow; K], and Day 2 of pregnancy [L]) ofWT mice. The ampulla is characterized by a thin smooth muscle layer, a thin layer of subepithelial connective tissue and long longitudinal mucosal foldsdisplaying a high percentage of ciliated cells in the epithelium (A–F). The isthmus shows a thick smooth muscle layer, a very distinct layer of subepithelialconnective tissue, and circular mucosal folds with mainly secretory cells—single ciliated cells are only seen in the depth between the folds (arrow; G–L).During estrus, the number of secretory cells in the ampulla increases, and the secretory cells start to arise over the level of the ciliated cells. The activity ofsecretory cells as seen by cell protrusions (arrows) in the ampulla increases from metestrus (C) and diestrus (D) to the first day of pregnancy (E). On thesecond day of pregnancy, many emptied secretory cells are visible in the ampulla (arrowhead; F). In the isthmus, however, no changes are seen in theepithelial morphology during the estrous cycle. In the isthmus, a continuous high secretory activity is found during estrus (seen as accumulation of fluid inthe lumen, arrowhead; H), during metestrus, and during pregnancy. Bar¼ 20 lm.

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rendering particle tracking impossible. Application of ATPtended to result in a decrease of PTS (92 6 14 lm/sec vs. 776 11 lm/sec), whereas atropine exerted no influence.Generally, contraction of the musculature was more pro-nounced in pregnant mice as compared to unmated mice,especially on Day 2 of pregnancy. Oviducts from pregnantmice also showed augmented contraction after exposure toATP.

CBF of the Oviduct in WT Mice

Because of the location of ciliated cells between the circularfolds and the rhythmic contractions of the muscle layer in theisthmus region, CBF was measured only in the ampulla.Ciliated cells showed an average beat frequency of 21 6 1 Hz(Figs. 4C and 8A, inset, and Supplemental Movie S3), whichsignificantly (P , 0.05) slowed down within 2 min in responseto ATP (10�4 M; 21 6 2 vs. 20 6 2 Hz) (Fig. 8A, inset).

PTS and CBF in the Trachea in Comparison to the Ampullaof WT Mice

In contrast to the ampulla (basal PTS, 71 6 7 lm/sec), thebasal PTS of the trachea was low (21 6 5 lm/sec) (Figs. 4D,inset, and 9, A and C, and Supplemental Movie S4) and wassignificantly stimulated (up to 50 6 5 lm/sec) by ATP (10�4

M) (Fig. 9B). Application of atropine (10�6 M) did not affectPTS (Fig. 9A). Tracheal CBF was significantly stimulated (76 1 Hz vs. 21 6 2 Hz) by ATP (10�4 M) (Fig. 9A), but thebaseline frequency of 7 6 1 Hz was significantly lower ascompared with the oviduct (21 6 2 Hz) (Figs. 4D and 9D andSupplemental Movie S5).

FIG. 2. Localization of ciliated cells by immunohistochemistry andscanning-electron microscopy of ampulla and isthmus. A and B) Ciliatedcells as visualized by b-tubulin immunoreactivity (arrow) are numerous inthe ampulla (A) but are restricted to mucosal niches in the isthmic region(B). C and D) Secretory cells in ampulla (C) and isthmus (D) arecharacterized by the appearance of microvilli (arrows) on their cellsurface. In the ampulla, ciliated cells predominate (C), whereas in theisthmus, mainly secretory cells are visible (D). Bar¼ 20 lm (A and B), 10lm (C), and 5 lm (D).

FIG. 3. Relative frequency of ciliated cells in ampulla and isthmusdepending on estrous cycle stage and in early pregnancy. In the ampulla,the percentage of ciliated cells is significantly higher as compared to thatin the isthmus during all estrous cycle stages and early pregnancy. In theampulla, a higher relative frequency of ciliated cells is found during estrus(Estr) as compared to Days 1 and 2 of pregnancy. A significant higherrelative frequency of ciliated cells is seen in the isthmus at estruscompared to the other cyclic stages and the first day of pregnancy. Dataare displayed as the mean 6 SEM. Statistical analysis was carried out byKruskal-Wallis test, followed by Mann-Whitney test. Di, diestrus; Met,metestrus; Pro, proestrus. *P , 0.05.

FIG. 4. Videomicroscopic analyses of particle transport and ciliarybeating. A) Ampulla. Particles are mainly transported in the depth betweenthe folds (see Supplemental Movie S1). B) Isthmus. Nearly no particletransport is observed. Particles settle down in the depth between the folds.Strong muscular contractions are regularly seen (see Supplemental MovieS2). C) Ampulla. Ciliated cells of the ampulla beat at a high rate of 21 Hz(see Supplemental Movie S3). D) Trachea. The basal CBF in the trachea (7Hz) is 3-fold lower as compared to that in the ampulla (21 Hz; seeSupplemental Movie S5). Ciliated cells are always arranged in groups. ThePTS in the trachea (inset) is more than 3-fold lower (22 lm/sec) ascompared to that in the ampulla (71 lm/sec; see Supplemental Movie S4).Bar¼ 100 lm (A and B) and 5 lm (C and D).

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Messenger RNA Expression of Chrm1 and Chrm3 in theOviductal Epithelium

Reverse transcription and PCR of total RNA isolated fromampulla, isthmus, and uterotubal junction revealed expressionof all Chrm subtypes (Chrm1 to Chrm5) in each segment of theoviduct. Electrophoresis of PCR products derived from RT-PCR with primers Chrm1i and Chrm3i showed doubled bands.Sequencing confirmed the presence of two transcript variantsfor Chrm1. In microdissected samples of oviductal epithelium,Chrm1 and Chrm3, but not Chrm2, were detected (Fig. 10).Amplification products were not obtained without reversetranscription. In view of the absence of Chrm4 and Chrm5from other known ciliated epithelia and the comparably weaksignals obtained in grossly dissected oviductal pieces, theirexpression was not specifically investigated in microdissectedepithelium.

DISCUSSION

The present study provides the first data, to our knowledge,on CBF and cilia-driven particle transport in the murine

oviduct, demonstrating high basal activity throughout all cyclicstages and early pregnancy that corresponds to a level that canbe achieved in the trachea only by stimulation. The high CBFvalue of 21 Hz that we recorded in the ampulla is at the upperrange of what has been reported for mammalian cilia in theoviduct. In the human oviduct, a wide range of frequencies(between 5 and 20 Hz, but mostly ,10 Hz) has been reported,with the differences being ascribed to the various methodsemployed for assessment [19–21]. In view of this methodo-logical discussion, it is important to note that the basal CBFvalues we record with our system in the murine trachea (7-10Hz) [7, 8] (present study) are well supported by studies fromother groups utilizing different setups and other systems (10–11 Hz) [22–24]. Further validation of the system derives fromthe finding that CBF and PTS changed in parallel uponstimulation of the murine trachea, because mucus/fluid-propelling velocity is linearly dependent on CBF [25].

Stimulus-induced modulation of CBF and cilia-drivenparticle transport was remarkably low in the present study, inthat only ATP at 10�4 M reduced CBF by 1 Hz (from 21 to 20Hz, corresponding to 5%) and particle transport by 15 lm/sec

FIG. 5. Measurement of PTS in the ampulla (A) and in the isthmus (B). In the ampulla, the average PTS is 65 6 4 lm/sec. In the isthmus, only 1 of 27samples displayed measurable particle transport. The graph depicts PTS from this single experiment. Data are displayed as the mean 6 SEM. The x-axisdepicts time after dissection.

FIG. 6. Basal PTS in the oviduct of cycling (A), early pregnant (B), and Chrm-deficient (C) mice. Box plots depict the median and quartiles 0–100; databeyond 3-fold the SD are represented by circles. Kruskal-Wallis test revealed no significant differences between groups. Di, diestrus; Estr, estrus; Met,metestrus; Pro, proestrus.

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(from 92 to 77 lm/s, corresponding to 16%) within 2 min. Anopposite effect of ATP (i.e., an ATP-induced increase in CBFby ;80% from a baseline of 11.35 Hz) has been reported forhamster oviductal ciliated cells grown in culture as amonolayer for 5 days [12, 26]. Although species differencesand the use of freshly excised oviducts versus 5 days of cellculture do not allow direct comparison between these twoapproaches, it is noteworthy that in both cases, CBF reached 20Hz after ATP application. A low acute dynamic range similarto that observed in the present study has also been reported forCBF of cultured or explanted oviductal cells from several otherspecies. Bovine ciliated cells respond to mifepristone, aprogesterone receptor blocker, and to progesterone itself witha 3% and 11% inhibition, respectively, of CBF within 15–90

min [6]; interleukin-6 reduces human oviductal CBF by 0.62Hz (baseline, 5.5–6 Hz) within 30 min [27]; and 4-phorbol12,13-didecanoate, a synthetic activator of the transientreceptor potential cation channel subfamily V member 4,increases CBF of hamster oviductal cells by 15% (baseline, 12Hz) after 5-10 min [28].

This low dynamic regulatory range in the oviduct is inmarked contrast to both the extent and the direction of the ATPeffect in the murine trachea, where CBF increased by 3-fold(from 7 to 21 Hz) and PTS by 2.5-fold (from 21 to 50 lm/sec),confirming our previous data of a basal CBF of 5-10 Hz thatwas increased by appropriate stimuli up to 30 Hz [7, 8]. Insteadof ATP, 5-HT induces a strong increase in CBF from basalrange of 19–22 Hz up to 28 Hz in rat brain ependymal cells[29] and from 9 to 17 Hz in the mouse trachea [8] but, again,not in the mouse oviduct. Similarly, CHRM agonists(muscarine and ACh) markedly stimulated CBF and PTS inthe mouse trachea [7] (present study) but were ineffective inthe mouse oviduct. In view of the facts that 1) many epithelia,including tracheal and oviductal, produce ACh [13, 14]; 2)gene deficiency of Chrm3 results in reduced tracheal basalCBF and PTS [7]; and 3) Chrm3 is expressed by the oviductalepithelium (present study), we considered the possibility thatthe fixed high basal CBF we observed in the oviductal ampullamight result from a continuous local release of ACh. However,neither administration of the general CHRM antagonistatropine nor gene deficiency of any of the Chrm subtypeshad any impact upon CBF and particle transport. In the mousetrachea, Chrm1 also drives PTS [7], and we found this receptoralso being expressed by the oviductal epithelium. Again,however, neither its deficiency nor its stimulation by thenatural agonist ACh had an acute impact on oviductal CBF andparticle transport. Thus, although the oviductal epitheliumshares the Chrm expression pattern with that of the trachealepithelium (Chrm1 and Chrm3), these cholinergic receptors arelinked to different regulatory pathways in these mucosalsurfaces.

In mammals, the only other system in which we are awareof a similarly fixed high basal CBF (22 Hz at 288C–348C) is themurine small intrapulmonary airways [30]. This has been

FIG. 7. PTS in the oviduct of cycling, early pregnant, and Chrm-deficientmice in response to ATP and atropine. ATP leads to a significant decrease2 min after the administration in WT and Chrm3-deficient mice, whereasatropine was without effect (Friedman test, followed by Wilcoxon test, *P, 0.05). Data are displayed as the mean 6 SEM.

FIG. 8. PTS in the oviduct of WT mice after application of muscarine (A) and ACh and 5-HT (B). Data are displayed as the mean 6 SEM. Statisticalanalysis was carried out by Friedman test, which showed no statistically significant difference at any of the time points examined. Inset in A showsmeasurement of CBF in the ampulla. Basal CBF decreases significantly in response to ATP (Wilcoxon test; *P , 0.05). Data are displayed as the mean 6

SEM.

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termed ‘‘autonomous activity,’’ and it has been proposed toserve for sustained optimal mucociliary clearance of inhaledparticles, such as bacteria [30]. In the present study, a high rateof directed particle transport could be directly visualized.Accordingly, along the same line of argumentation as putforward for the small murine airways, we propose thatoviductal autonomous ciliary activity serves to maintainoptimal clearance of tubal content throughout all estrous cyclestages and early pregnancy.

This independence from estrous cycle stage indicates theimportance of tubal clearance beyond gamete transport, such asin clearance of tubal secretions and ascending bacteria. Indeed,mean bacterial counts in the uterine horn are significantlyhigher than in the contiguous oviduct following intrauterineinjection of a standard inoculum of Escherichia coli bymicropuncture technique in BALB/c mice, demonstrating thepresence of mechanisms hindering bacterial ascent [31].

We found only little dynamic regulation of CBF and particletransport upon pharmacological stimulation in the ampulla, butwe noted a striking difference in particle transport betweenvarious anatomical sites of the oviduct (i.e., ampulla andisthmus). Effective particle transport was practically undetect-able in the opened isthmus. Similarly, early qualitative

observations on the transport of 15-lm particles and stainedlycopodium spores on the surface of opened oviductalsegments of rat and guinea pig revealed effective transport inthe ampulla but not in the isthmus [32]. In larger animals (cowand sheep), however, isthmic transport was noted [32]. Rat,guinea pig [32], and mouse (present study) oviducts share thestructural feature of a richly ciliated ampulla (;60% of cellsare ciliated in the mouse) and a marked paucity (mouse,;10%) of ciliated cells in the isthmus. This easily explains theabsence of surface particle transport on an opened isthmus. Itcannot be interpreted, however, as the absence of cilia-driventransport in the closed isthmus in situ. Because the ampulla andisthmus are segments of a contiguous tube, it is physicallyimpossible to generate a flow in an upstream segment (i.e.,ampulla) without causing flow in downstream segments (i.e.,isthmus) as well, even if this downstream segment by itselfdoes not, or does not significantly, contribute to the generatingforce. Moreover, the fluid volume contained within the closedisthmus with its folded mucosa is much smaller than thatoverlaying the opened and largely unfolded isthmic mucosa, sothe driving force generated by the relatively small number ofciliated cells may suffice to create or sustain directed flowunder in situ conditions.

FIG. 9. Comparison of tracheal and oviductal PTS and CBF. A) PTS in the trachea. Basal PTS is low, is significantly increased by ATP, and is unaffected byatropine (Wilcoxon test, *P , 0.05). B) CBF in the trachea. Basal CBF is low and is significantly increased by ATP (Wilcoxon test, *P , 0.05). C) Basal PTSin the oviductal ampulla is significantly higher than in the trachea (Mann-Whitney test, ***P , 0.001). D) Basal CBF is significantly higher in the oviductalampulla than in the trachea (Mann-Whitney test, ***P , 0.001). Data in A and B are displayed as the mean 6 SEM; box plots in C and D depict themedian and quartiles 0–100.

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The cellular composition of the oviductal epitheliumchanges with estrous cycle stage in many species (goat [33],pig [34], cow [35], dog [36], and human [37]). Accordingly,we noted changes in the relative number of ciliated cellsdepending on estrous cycle stage and early pregnancy inC57BL/6 mice, albeit they were not as pronounced as thosereported for the ICR mouse strain by Morita et al. [38]. Thoseauthors observed 76% and 90% frequency of nonciliated cellsin the ampulla in estrus and metestrus, respectively, whereaswe rather continuously counted approximately 30% forampullar nonciliated cells. This striking contrast may be dueto strain differences, but it is also conceivable that the ampullarsegments investigated by Morita et al. [38] were taken closer tothe isthmus than our samples. The major estrous cycle stage-dependent change we observed was a 4-fold increase in therelative frequency of ciliated cells in the isthmus from proestrus(6%) to estrus (24%) with subsequent return to 10%, indicatingparticularly well-sustained transport capacity at ovum pickup.In the ampulla, significant changes occurred between estrus(72% ciliated cells) and the first two days of pregnancy (62% ateach day). The concomitant relative increase in secretory cellsis consistent with a high tubal secretory capacity in the firstdays of pregnancy [39].

This slight reduction in the frequency of ciliated cells inearly pregnancy had no impact upon CBF and particletransport, indicating that maximum transport speed can beachieved by a relative ciliated cell frequency of 62% or evenless. This assumption is supported by data obtained in parallelat the mouse trachea, where ciliated cells account for 30% only[7] and, upon stimulation, a CBF of 21 Hz and a transportspeed of approximately 50 lm/sec were reached, which arewithin the range of transport speeds seen in the ampulla.Hence, a sufficient cellular reserve to maintain the high basalPTS in the mouse oviductal ampulla apparently exists evenunder conditions of epithelial remodeling.

In conclusion, the present study demonstrates a fixed highCBF and cilia-driven particle transport (autonomous activity)in the murine oviduct that, despite the presence of CHRMsubtypes, is neither driven by endogenous ACh nor can befurther stimulated by muscarinic agonists. We propose that thisoviductal autonomous ciliary activity serves to maintainoptimal clearance of tubal content throughout all estrous cyclestages and early pregnancy. The high relative frequency ofciliated cells in the ampulla and the potent particle transportcapacity of ampullar segments, together with the paucity ofciliated cells in the isthmus and the inability of the openedisthmic segment to generate directed particle transport, definethe ampulla as the motor of this tubal clearance mechanism.

ACKNOWLEDGMENT

We thank Dr. Jurgen Wess (National Institute of Diabetes andDigestive and Kidney Diseases, Bethesda, MD) for providing the Chrm-deficient mice. The excellent technical assistance of Mrs. Petra Faul-hammer, Mrs. Petra Hartmann, and Mr. Martin Bodenbenner (Institute ofAnatomy and Cell Biology of the University of Giessen) and of Mrs.Kathrin Wolf-Hofmann, Mrs. Susanne Schubert-Porth, and Mrs. JuttaDern-Wieloch (Institute of Veterinary Anatomy, Histology and Embryologyof the University of Giessen) is gratefully acknowledged.

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