Abstract Background The oviduct (or fallopian tube) plays a functional role in the transportation of gametes and early-stage embryos, serving as a site for fertilization. Any aberrations from the normal functioning of fallopian tube epithelium may lead to diseases, including but not limited to infertility, ectopic pregnancy, and carcinoma. However, little is known about the mechanisms governing oviductal epithelial homeostasis between secretory cells and ciliated cells by miRNAs. Results In this study, we found that the expression levels of miR-34b/c and miR-449a/b/c were positively correlated with the number of ciliated cells. Using the miR-34b/c^−/−; miR-449 ^−/− double knockout (miR-dKO) mouse model, we found that adult female miR-dKO mice were infertile, owing to reduced ciliated cells and increased secretory cells, which hindered oocyte pickup. Transcriptomic analysis revealed Wnt/β-catenin signaling over-activation in the oviductal epithelium of the miR-dKO mice, with Dvl2 as a target of miR-34/449. The aberrant ciliated cell differentiation was completely rescued with the treatment of the Wnt/β-catenin signaling pathway inhibitors and partially rescued with the knockdown of Dvl2, using the oviductal epithelial organoid culture model. Conclusions In summary, we discovered that ablation of miR-34/449 led to hyperactivation of the Wnt/β-catenin signaling pathway, which resulted in the differentiation impairment of ciliated cells, thus resulting in infertility in the mice. This study revealed a novel mechanism describing how miR-34/449 affects oviductal ciliated cell differentiation and oviductal epithelial homeostasis through the Wnt/β-catenin signaling pathway and finally affects fertility. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-025-01468-w. Keywords: miR-34/449, Oviduct, Wnt/β-catenin, Organoid, Dvl2 Introduction Mouse oviduct (analogous to the fallopian tube in humans) stands as a cornerstone of the female reproductive system, serving as a vital conduit bridging the ovaries to the uterus [[34]1–[35]3]. This intricate structure is subdivided into distinct segments, including the infundibulum, the ampulla, the isthmus, and the uterotubal junction [[36]4]. The epithelium lining the oviduct primarily comprises secretory and ciliated cells [[37]5]. Ciliated cells are responsible for the movement of eggs and embryos through the oviducts toward the uterus [[38]6], while secretory cells produce substances that facilitate the transport and nourishment of the eggs and early-stage embryos [[39]7]. Together, these cell types contribute to maintaining the homeostasis and renewal of the epithelium. An in vivo genetic cell lineage tracing experiment found that oviductal secretory cells possess the remarkable ability to self-renew and subsequently differentiate into ciliated cells. Moreover, an intermediate cell state expressed both secretory cell-specific (PAX8) and ciliated cell-specific (Ac-α-tubulin) markers during the differentiation process from secretory cell to ciliated cell [[40]8]. Other experiments demonstrated that cells co-expressing PAX8 and FOXJ1 (another ciliated cell marker), or KRT7 (a secretory cell marker) and CAPS (a ciliated cell marker), represented as an intermediate cell state within the fallopian tube, as revealed by single-cell transcriptome analysis [[41]9, [42]10]. Taken together, secretory cells exhibit biopotency: it can either maintain their numbers through proliferation or differentiate into intermediate cells and subsequently into ciliated cells, which play a crucial role in oviductal epithelial homeostasis [[43]11]. Notwithstanding the pivotal importance of oviductal epithelial homeostasis in both reproductive function and disease, the intricate mechanisms orchestrating the homeostasis remain largely unknown. The ciliated cells in the oviductal epithelium are classified as multiciliated cells (MCCs). MCCs are a type of terminally differentiated epithelial cell with multiple motile cilia on the luminal surface, which are primarily present in the respiratory tract, ventricular ependyma, oviduct, and efferent ductule [[44]12, [45]13]. Previous studies have shown that two miRNA clusters, miR-34b/c and miR-449a/b/c, play a crucial role in the development and function of motile cilia in various organs, including respiratory tract, brain ventricle, efferent ductules, and oviduct [[46]14–[47]17]. The miR-34b/c cluster on chromosome 9 encodes two miRNAs (miR-34b and miR-34c), and the miR-449 cluster on chromosome 13 encodes three (miR-449a, miR-449b, and miR-449c). All five miRNAs have the same seed sequence, which is also shared by another miRNA, miR-34a, which is encoded by miR-34a loci on chromosome 4. Therefore, these six miRNAs form a functionally related miRNA family. Interestingly, studies have shown that the expression levels of miR-34b and miR-34c are elevated (while the expression of miR-34a remains unchanged) in miR-449a/b/c ^−/− mice, and similarly, the expression levels of miR-449a, miR-449b and miR-449c are increased in miR-34b/c ^−/− mice. Moreover, miR-449a/b/c and miR-34b/c regulate the same target genes, suggesting functional redundancy between miR-449a/b/c and miR-34b/c [[48]16, [49]18]. Particularly, studies have demonstrated that miR-34b/c^−/−;miR-449a/b/c^−/− knockout in mice results in abnormal ciliogenesis, with a significant decrease in the number of ciliated cells and motility of cilia in the oviduct [[50]16, [51]17]. However, whether miR-34/449 (referring to miR-34b/c and miR-449a/b/c in this study) regulates the differentiation of ciliated cells and the oviductal epithelial homeostasis, as well as the mechanism underlying this process, remains to be further investigated. In this study, by using both the miR-34/449 double knockout (miR-dKO) mouse model and the oviductal epithelial organoid model, we discovered that the deletion of miR-34b/c and miR-449a/b/c may cause hyperactivation of the Wnt/β-catenin signaling pathway by upregulating Dvl2, causing over-proliferation of the oviductal secretory cells and the aberrant differentiation of the ciliated cells, which leads to a significant reduction in the number of ciliated cells in the oviductal epithelium, thus resulting in infertility in the mice. This study revealed a new mechanism whereby miR-34/449 regulates the differentiation of ciliated cells and epithelial homeostasis in the oviduct by targeting the Wnt/β-catenin signaling pathway. Methods and materials Animals All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the Shanghai Jiao Tong University School of Medicine. The miR-34b/c^−/−; miR-449^−/− double knock-out (miR-dKO) mice were generated as described previously [[52]16]. All control (CON) mice were littermates of the analyzed miR-dKO mice. All mice used in the experiment were maintained in the C57BL/6 J background. Superovulation of female mice Three-week-old female miR-dKO and control mice were injected intraperitoneally with 5 IU pregnant mare’s serum gonadotropin (PMSG) (200 µL of a 25 IU/mL solution in 0.9% NaCl), followed by an injection of 5 IU human chorionic gonadotropin (hCG) (200 µL of a 25 IU/mL solution in 0.9% NaCl) 48 h after PMSG. Mice were then euthanized 16 h after hCG injection, after which their ovaries and oviducts were processed for further histological analysis. Total RNA isolation and qRT-PCR analysis for mRNAs Total RNA was isolated from tissues and cultured cells using TRIzol reagent (Invitrogen, catalog no.15596026CN, USA) according to the manufacturer’s instructions. For reverse transcription, cDNA was prepared from 1 μg of RNA using PrimeScript RT Master Mix (Takara, catalog no. RR306A, Japan). The quantitative real-time PCR reaction was prepared using the TB Green Premix Ex Taq II (Takara, catalog no. RR820A, Japan) following the manufacturer’s protocol and performed using an Applied Biosystems 7500 instrument (ABI, USA). Relative quantification of the mRNA levels was calculated using the threshold cycle (CT) method 2^−∆∆Ct with Gapdh as the endogenous control. The primer sequences used in this study are listed in Supplementary Table [53]S1 and were synthesized by Sangon Biotech (Shanghai, China). Small RNA isolation and qRT-PCR analysis for miRNAs Small RNAs were isolated from oviducts or 293 T cells using the mirVana miRNA isolation kit (Ambion, catalog no. AM1561, USA) according to the manufacturer’s instructions. Quantification of the miRNA was performed with a bulge-loop miRNA qRT-PCR starter kit (RiboBio, catalog no. [54]C10211, China). Briefly, miRNA-cDNAs were transversed using RT primers modified with the bulge-loop structure. Each individual miRNA was amplified utilizing both reverse and forward transcription primers, with miRNA-cDNAs serving as templates. The forward primer corresponds to the miRNA sequence itself, while the reverse primer is designed based on the specific bulge-loop structure, enabling the differentiation of individual miRNAs within each cluster. The levels of miRNA were normalized to U6 snRNA (small nuclear RNA). The expression levels of the indicated genes were normalized to the endogenous or exogenous reference control using the 2^−ΔΔCt method. The Sequences of the primers used for qRT-PCR to detect miRNAs in this study are listed in Supplementary Table [55]S2. Histology and immunofluorescent staining For histology, oviduct tissues were dissected under a dissection microscope and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C followed by dehydration and paraffin embedding. Paraffin Sects. (4 μm) were cut using a microtome and stained with hematoxylin and eosin after deparaffinization and rehydration. For immunofluorescent staining, tissue samples were fixed in 4% paraformaldehyde in PBS overnight at 4 °C and then dehydrated in a graded series of sucrose solutions (from 10 to 30%) and embedded with optimal cutting temperature (OCT) compound (1:1) (Tissue-Tek, catalog no. 4583, USA). Cryosections (6 μm) were cut at − 20 °C, and the sections were washed twice in PBS. After blocking in PBSTB (0.1% Triton X-100, 1% bovine serum albumin in PBS) at room temperature for 1 h (h), the slides were incubated with primary antibodies at 4 °C overnight. The primary antibodies used included anti–acetylated-α-tubulin antibodies (Sigma, catalog no. T6793; 1:500), anti-PAX8 antibodies (ProteinTech, catalog no.10336–1-AP; 1:200), anti-Ki67 antibodies (Abcam, catalog no. ab15580, catalog no. ab156956; 1:200). After washing three times with cold PBST (0.1% Triton X-100 in PBS), the slides were incubated with a secondary antibody (Yeasen, Alexa Fluor 488 goat anti-mouse, catalog no. 33906ES; Alexa Fluor 594 conjugate goat anti-rabbit, catalog no. 33112ES; Alexa Fluor 488 conjugate donkey anti-rat, catalog no. 34406ES; 1:400, China) at room temperature for 1 h. Slides were then mounted with a mounting medium containing 4’, 6-diamidino-2-phenylindole (DAPI) (Yeasen, catalog no. 36308ES, China). Images were taken with an epifluorescence microscope (Olympus, Japan). Separation of oviductal epithelium To obtain enriched oviductal epithelial cells, we adopted a previously reported method with slight modifications to separate oviductal epithelium [[56]17]. In brief, the oviducts were dissected from adult wild-type (WT), control, and the miR-dKO female mice and then transferred to 1 × Hanks’ balanced salt solution (HBSS) solution (HyClone, catalog no. SH3058801, USA) containing 1% trypsin (Sigma, catalog no. T-4799, USA), followed by incubation at 4 °C for 90 min and then at 22 °C for 30 min. After incubation, the oviducts were rinsed in 20% fetal bovine serum in 1 × HBSS solution (HyClone, catalog no. SH30588, USA) and then treated with 0.1% deoxyribonuclease I (Sigma, catalog no. DN25, USA) in 1 × HBSS. Epithelial and stromal layers were separated by gentle suction of the oviducts through a glass capillary pipette under a stereomicroscope. The separated epithelial and stromal layers were washed several times with PBS and then collected for RNA extraction and RNA-seq. Protein preparation and western blotting analysis Samples were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, catalog no. 89901, USA) containing protease inhibitor cocktail (Roche Applied Science, catalog no. 11836170001, Switzerland) on ice for around 30 min for total protein extraction. The proteins were collected in the supernatant after centrifugation at 14,000 × g for 10 min at 4 °C, and the protein concentrations were determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, catalog no. 23227, USA). The protein samples were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes (Millipore, catalog no. IPFL00010, USA). The membranes were blocked in 5% BSA for 1 h at room temperature, followed by incubation in primary antibodies recognizing FOXJ1(Invitrogen, catalog no.14–9965-82; 1:1000), PAX8 (ProteinTech, catalog no. 10336–1-AP; 1:1000), β-catenin (CST, catalog no. 8480S; 1:1000), DVL-2 (Santa Cruz, sc-8026; 1:500) and GAPDH (Santa Cruz, catalog no. sc-365062; 1:200). And then incubated with horseradish peroxidase-conjugated secondary antibodies (CST, catalog no. 7076S, 1:5000, USA). The immunoreactive bands were visualized using a chemiluminescence image system (Millipore, catalog no. WBKLS0100, USA). Transmission electron microscopy analysis Oviducts from mice aged 1-week-old, 2-week-old and 3-week-old were immersed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 °C and postfixed in 1% osmium tetroxide for 2 h at 4 °C. After dehydration, the samples were embedded in Epon 618 (TAAB Laboratories Equipment, UK) and cut to ultrathin Sects. (70–90 nm), followed by staining with uranyl acetate and lead citrate. The ultrastructure of the oviduct was observed using a transmission electron microscope (Philips CM-120, the Netherlands). TUNEL apoptosis assay Apoptotic cells in the cryosections were detected using a TUNEL assay kit (Ribobio, catalog no. C11026-1, China) according to the manufacturer's instructions. Briefly, oviduct cryosections were fixed with 4% paraformaldehyde at 37 °C for 15 min and then blocked using a blocking buffer. Subsequently, the sections were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. The sections were incubated with a TUNEL reaction mixture for 1 h at 37 °C. DAPI was used to counterstain the nuclei, and the numbers of TUNEL-positive cells were recorded under a fluorescence microscope. Plasmids and transfection Total RNA extracted from oviduct tissues using TRIzol reagent (Invitrogen, catalog no.15596026CN, USA) was used for cDNA synthesis by reverse-transcription PCR. The obtained cDNA was used as a template to amplify the coding sequence (CDS) of genes of interest in the course of subsequent cloning. Dvl2 was amplified by PCR and subcloned into pLVX-puro. The pGIPZ vector was used to construct shRNAs targeting Dvl2 and the empty vector was used as the negative control. Stable cell lines were obtained after selection with 2 μg/mL puromycin for two weeks. The plasmids were transfected into 293 T cells using Lipofectamine 3000 (Invitrogen, catalog no. L3000015, USA) according to the manufacturer's instructions. The shRNA targeting sequences are as follows: Dvl2-shRNA1: 5’- CTGTGAGAGTTACCTAGTTAA -3’. Dvl2-shRNA2: 5’- ACCCATCTTGAGGCCACATTG -3’. Dual-luciferase reporter assay For miR-34b/c, miR449 target gene validation, the 3’UTR (untranslated region) of Dvl2 was amplified by PrimeSTAR® Max DNA Polymerase (Takara, catalog no. R045A, Japan) from mouse genomic DNA using PCR. After being digested with Xho I (NEB, catalog no. R0146S, USA) and Not I (NEB, catalog no. R3189S, USA) restriction enzymes, the PCR products were inserted into the pmiR-REPORT vector (RoboBio, catalog no. R30012.3, China). The pmiR-Dvl2-3’UTR-Mutant plasmid containing the mutated Dvl2-3’UTR binding site “GCCCACC” was generated using a QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, catalog no. 210518, USA). The 293 T cells were cultured in 24-well plates and were co-transfected with 200 ng pmiR-Dvl2-3’UTR or pmiR-Dvl2-3’UTR-Mutant plasmids and 5 pmol miR-34b/c, miR-449 mimics (RiboBio, China) using Lipofectamine 3000 reagent (Invitrogen, catalog no. L3000015, USA). A miRNA mimic control (RiboBio, China) was used as a negative control. The Dual-Luciferase Reporter Assay System (Promega, catalog no. E1910, USA) was used to measure the activities of firefly luciferase and renilla luciferase in the cell lysates collected 24 h post-transfection, according to the manufacturer’s instructions. TOPflash/FOPflash reporter assay For the Wnt/β-catenin reporter assay in primary oviductal epithelial cells, primary oviductal epithelial cells were transfected with TOPflash (Addgene, catalog no. 12456, USA) or FOPflash (Addgene, catalog no. 12457, USA), and pRL-TK (Promega, catalog no. E2241, USA). The Dual-Luciferase Reporter Assay System (Promega, catalog no. E1910, USA) was used to measure the activities of firefly luciferase and renilla luciferase using the cell lysates following the manufacturer’s protocol. The activity of firefly luciferase was normalized to that of renilla luciferase. For detecting the Wnt/β-catenin reporter assay in oviduct organoids, primary oviductal epithelial cells were co-transfected with TOPflash or FOPflash. The transfected cells were subsequently used to establish organoid cultures. After one month of culture, organoids were harvested, and luciferase activity was measured to evaluate reporter activity. Primary oviductal epithelial cell culture Mouse oviductal epithelial cells were isolated from either 3-week-old control or miR-dKO mice in dissection solution (PBS containing 2% fetal bovine serum (FBS) and 100 IU/ml of Penicillin and 100 ug/ml of Streptomycin). Oviducts were then broken into 1 mm pieces with forceps before being incubated in collagenase Ⅰ (5 mg/ml) and DNase I (5 U/100 μl) in a dissection solution for 35 min at 37 °C. Oviducts were then centrifuged for 5 min at 250 g, resuspended in 100 μl 0.25% trypsin, and incubated for 5 min at 37 °C. Oviducts were again centrifuged for 5 min at 250 g and resuspended in 200 μl dissection solution. The resulting oviducts and cell suspensions were passed through an 18 G needle 10 times to release epithelial cells and then through a 40 μm cell strainer. Collected cells were resuspended in 100 μl 2D culture media [DMEM/Ham’s F12 (Gibco, catalog no. 11320033, USA), 15 mM HEPES (Gibco, catalog no. 15630080, USA), 100 U/ml penicillin (Gibco, catalog no. 15140122, USA), 100 μg/ml streptomycin (Gibco, catalog no. 15140122, USA), 1X GlutaMAX (Gibco, catalog no. 35050–061, USA), 5% FBS (Wisent Bioproducts, catalog no. 090–150, Canada), 1X insulin-transferrin-selenium (Gibco, catalog no. 41400045, USA), 25 ng/ml hEGF (Peprotech, catalog no. AF-100–15-500, USA), 30 μg/ml bovine pituitary extract (Gibco, catalog no. 13028014, USA)]. Oviductal epithelial organoid culture and passage Epithelial cells were grown in a 37 °C humidified incubator with 5% CO[2] until 70% of cells were confluent (2–5 days). Epithelial cells were then seeded at a density of 25,000 cells per 50 μl drop of matrigel in 24-well uncoated dishes which were allowed to set inverted for 30 min in a 37 °C humidified incubator with 5% CO[2] before being submerged in BET organoid medium [DMEM/F12 advance media (Gibco, catalog no. LS12634028, USA) with HEPES (Gibco, catalog no. 15630080, USA), 1X GlutaMAX (Gibco, catalog no. 35050–061, USA), 1X B27 (Gibco, catalog no. 17504044, USA), 0.1 μg/ml hEGF (PeproTech, catalog no. AF-100–15-500, USA), 0.5μΜ TGFBR1 Kinase Inhibitor IV (BioVision, SB431542, catalog no. 1674–1, USA)]. For the first four days of culture after seeding and passaging, 10 μM of ROCK inhibitor Y-27632 (Sigma, catalog no. Y0503, USA) was added. Organoids were passaged as described previously [[57]19] every 7–14 days and differentiation was induced by the addition of 1 μM selective γ-secretase inhibitor Y0-01027 (DBZ) (Calbiochem, catalog no. 209984–56-5, USA) to the organoid culture medium [[58]20]. For organoids treated with Wnt signaling pathway inhibitor XAV-939(MedChemExpress, catalog no. HY-15147, USA) or NSC668036 (MedChemExpress, catalog no. HY-117666, USA), XAV-939 or NSC668036 was added at a concentration of 3 µM or 10 µM into BET organoid medium and organoids were continuously exposed to the inhibitors over the culture period. Control organoids were cultured with BET organoid medium containing dimethyl sulfoxide (DMSO) as a vehicle. For organoids treated with Dvl2-shRNA or overexpression lentivirus, the lentivirus packaging system consisting of psPAX2 (Addgene) and PMD2.G (Addgene) was used to create virus particles. In brief, the plasmids were transfected into HEK293T packaging cells at 60% confluence using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. After an additional 48 h incubation, the supernatant was collected, filtered using a 0.45-µm filter (Millipore), and used to infect primary oviductal epithelial cells in the presence of 6 µg/mL polybrene (Yeasen, China). The resultant stable polyclonal populations of transduced cells were then selected with puromycin (Yeasen) for two weeks. And then, transduced cells were seeded for organoid culture. RNA-seq and bioinformatic analysis Oviductal epithelium from 3-week-old mice were harvested for RNA extraction. The quality of the RNA samples was analyzed by detection of RNA purity (A260/A280) and integrity. Complementary DNA library construction and quality inspection were performed using the above mRNA as the template. According to the effective concentration and data requirements, the library was pooled and then sequenced using the Illumina system (Illumina, USA). Then, differentially expressed gene (DEG) analysis and functional enrichment analysis, including GO and KEGG pathway analysis, were carried out. The target genes of miR-34b/c, and miR-449 were predicted by using Targetscan and miRDB. Gene set enrichment analysis Gene set enrichment analysis (GSEA) was performed to explore significant and underlying biological functions between the two groups. Gene sets were downloaded from the Molecular Signatures database, and GSEA was performed (version 4.0.3). Gene set permutations were performed 1,000 times to achieve a normalized enrichment score (NES) for each analysis. A nominal p < 0.05 and false discovery rate (FDR) < 0.05 were statistically significant. Statistical analysis The data are presented as the mean ± SD and were analyzed using GraphPad Prism 9 (GraphPad Inc., La Jolla, CA, USA). A Student’s t-test was used for comparison of two groups and a one-way ANOVA followed by Tukey’s post hoc multiple comparison correction was used for multi-group comparisons. An appropriate p < 0.05 was considered statistically significant. All data are representative of at least three independent experiments. Results The expression levels of miR-34/449 clusters in the oviductal epithelium are positively correlated with ciliated cell development The oviduct comprises epithelial, smooth muscle, and connective tissue layers. To investigate the expression of miR-34/449 in the oviduct, we first isolated and purified the epithelial layer from the stromal layer (consisting of smooth muscle cells and connective tissue) using enzymatic digestion and mechanical separation (Fig. [59]1A). The purity of the epithelial layer and stromal layer were identified using epithelial cell marker Ovgp1 and smooth muscle cell marker Des (Fig. [60]1B–C). Further analysis revealed that miR-34/449 were expressed primarily in the oviductal epithelium of the wild-type (WT) mice (Fig. [61]1D). Fig. 1. [62]Fig. 1 [63]Open in a new tab MiR-34/449 expression levels are positively correlated with the ciliated cell number in the oviductal epithelium. A Schematic diagram of the separation of the epithelial layer and stromal layer (consisting of smooth muscle cells and connective tissue) of a mouse’s oviduct. B–C Purity identification of the epithelial layer and stromal layer were tested using Ovgp1 as an epithelial cell marker and Des as a smooth muscle cell marker. D Relative miR-34/449 expression levels in the whole oviduct, epithelial layer, and stromal layer of the oviducts collected from the wild-type mice. E Representative immunofluorescence staining images of Ac-α-tubulin (indicating ciliated cells) in different developmental stages and different regions of the oviduct. “Distal” represents the infundibulum and ampulla regions of the oviduct; “Proximal” represents the isthmus and uterotubal junction regions of the oviduct. The box in the lower column shows the higher magnification of the box area in the picture. The white arrows indicate the ciliated cells. Scale bars: 50 μm. F Quantitative proportion of Ac-α-tubulin positive cells in the oviductal epithelial cells. G Relative miR-34/449 expression levels in different developmental stages of the oviducts. H Relative miR-34/449 expression levels in the proximal and distal regions of the oviduct. All the data are presented as the mean ± SD, n = 3. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001 Furthermore, utilizing immunofluorescence staining with the ciliated cell marker Ac-α-tubulin, we found that the numbers of ciliated cells in the proximal end (including the isthmus part and uterotubal junction part) of the oviduct peaked at two weeks post-birth, and this was followed by a plateau phase. In contrast, the numbers of ciliated cells in the distal end (including the infundibulum part and ampulla part) increased with the age of the mice, reaching a peak at 8 weeks post-birth, constituting approximately 80% of the oviductal epithelial cells. Notably, for the oviduct at the same developmental stage, the number of ciliated cells in the distal end consistently exceeded that in the proximal end, with the difference particularly pronounced at three weeks post-birth (Fig. [64]1E–F). In addition, the results demonstrated that the expression levels of miR-34/449 also increased with the age of the mice (Fig. [65]1G). Moreover, the expression levels of miR-34/449 were significantly higher in the distal end of the oviduct (Fig. [66]1H), which has more ciliated cells. These findings collectively suggest a positive correlation between the expression levels of miR-34/449 and the number of ciliated cells in the oviductal epithelium, highlighting the pivotal role of miR-34/449 in the differentiation and development of ciliated cells. Infertility in the miR-dKO female mice To probe further into the role of miR-34/449 in oviductal epithelial ciliated cell differentiation, we investigated the reproductive phenotypes of miR-dKO female mice. We first examined the expression of miR-34b/c and miR-449a/b/c in the oviducts and oviductal epithelia of the miR-dKO mice and control mice. We found that miR-34b/c and miR-449a/b/c were not expressed in the miR-dKO mice. However, ablation of miR-34b/c and miR-449a/b/c had no impact on the expression of miR-34a (another miRNA sharing the same seed sequence as miR-34/449) or miR-16 (a reference miRNA) (Fig. [67]S1A–B). The results suggest that the phenotype observed in the miR-dKO mice is attributed to the knockout of miR-34b/c and miR-449a/b/c. The miR-dKO female mice displayed complete infertility (Fig. [68]S1C). The cumulus-oocyte complexes retrieved from the control mice were found in the ampulla region of the oviduct, while those from the miR-dKO mice were noticeably trapped within the ovarian bursa, a unique anatomical structure that envelops the infundibulum and the ovary in rodents (Fig. [69]S1D). Based on the normal hormonal profiles and normal folliculogenesis of the miR-dKO female mice [[70]16], these findings suggest that the infertility phenotype observed in the miR-dKO females in the study is attributed to the failure of oocyte pickup by the oviduct, which is consistent with previous studies [[71]17]. The timely onset of decreased ciliated cells in the miR-dKO mouse oviducts In mice, the emergence of ciliated cells in the oviductal epithelium commences around postnatal day 4 (P4) and steadily increases with age [[72]8, [73]21]. To address the question of whether there is a reduction of ciliated cells in the oviductal epithelium of the miR-dKO mouse and, if so, when this reduction begins, we conducted immunofluorescence staining using a ciliated cell marker Ac-α-tubulin and a secretory cell marker PAX8 on the oviducts of the control and miR-dKO mice at different developmental stages. The results revealed that in the distal regions of the oviduct, a significant reduction in ciliated cell numbers and an increase in secretory cell numbers in the miR-dKO mice began as early as three weeks post-birth in the miR-dKO mice. Simultaneously, the number of intermediate cells (PAX8^+/Ac-α-tubulin^+) transitioning from secretory cells to ciliated cells significantly decreased, indicating impaired differentiation from secretory cells to ciliated cells, which contributed to the reduction in ciliated cell numbers (Fig. [74]2A–D). However, in the proximal regions of the oviduct, secretory cells, intermediated cells, and ciliated cells in the miR-dKO mice showed no significant differences compared with those of the control mice (Fig. [75]S2A–C). Further electron microscopy results supported these findings, revealing a decrease in ciliated cell numbers, reduced basal bodies, and disorganized ciliary arrangements in the oviductal epithelium of the miR-dKO mice at three weeks post-birth (Fig. [76]2E). These results collectively indicate that the reduction in ciliated cell numbers in the oviductal epithelium of miR-dKO mice significantly commences at three weeks post-birth. Interestingly, we observed a significant decline in intermediated cells and a concurrent increase in ciliated cells at week 8 in the distal regions of the oviduct in control mice (Fig. [77]2C–D). This result indicates that from week 5 to week 8 post-birth, most intermediated cells differentiated into ciliated cells, which accounts for the reduction in intermediated cells and increase in ciliated cells at week 8. Fig. 2. [78]Fig. 2 [79]Open in a new tab Decreased ciliated cells in the miR-dKO mouse oviduct appear at three weeks post-birth. A Representative co-immunofluorescence staining images of Ac-α-tubulin (indicating ciliated cells) and PAX8 (indicating secretory cells) in the distal region of the oviduct in the control (CON) and the miR-dKO (dKO) mice of different developmental stages. Scale bars: 50 μm. The box in the lower column shows the higher magnification of the box area in the picture. The white arrowheads indicate the ciliated cells (Ac-α-tubulin^+); The empty arrowheads indicate the secretory cells (PAX8^+); The white arrows indicate the intermediate cells (Ac-α-tubulin^+/PAX8^+). B, C, D Quantification of secretory cells, intermediate cells, and ciliated cells in the epithelium of the distal oviduct across different developmental stages. E Transmission electron microscopic images of the oviductal epithelium of the CON and the dKO mice across different developmental stages. Scale bars: 500 nm. The white dotted lines indicate the arrangement of the basal bodies. F Representative co-immunofluorescence staining images of PAX8 and Ki67 (indicating proliferating cells) in the oviductal epithelium of 3-week-old CON and dKO mice. Scale bars: 50 μm. The white arrows indicate the proliferating secretory cells (Ki67 + /PAX8^+). G Representative co-immunofluorescence staining images of Ac-α-tubulin and Ki67 in the oviductal epithelium of the CON and the dKO mice. Scale bars: 50 μm. The white arrows indicate the proliferating ciliated cells (Ki67 + / Ac-α-tubulin^+). H Quantification of Ki67^+ cells in the oviductal epithelium of the CON and the dKO mice. I Quantification of Ki67^+/PAX8^+ cells and Ki67^+/Ac-α-tubulin^+ cells in Ki67^−positive cells in the oviductal epithelium of the CON and the dKO mice. All the data are presented as the mean ± SD, n = 3. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001 In addition, through terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays, we discovered no significant differences between the miR-dKO mice and the control mice in the oviductal epithelium, suggesting that the reduction in ciliated cell numbers in the oviducts of the miR-dKO mice was not due to increased apoptosis (Fig. [80]S3A–C). Further examinations showed a significant increase in Ki67^+/PAX8^+ cells and a decrease in Ki67^+/Ac-α-tubulin^+ in the oviductal epithelium of the miR-dKO mice, in comparison with that of the control mice (Fig. [81]2F–I), indicating that the secretory cells in the miR-dKO mice oviducts were indeed in a state of vigorous proliferation, which prevented their exit from cell cycle and differentiation into ciliated cells and ultimately resulted in a reduction in the number of ciliated cells. Wnt/β-catenin signaling pathway hyperactivation in the oviductal epithelium of the miR-dKO mice To delve deeper into the reasons behind the ciliated cell differentiation impairment in the oviducts of the miR-dKO mice, we isolated the epithelial layers of the oviducts from three-week-old WT and miR-dKO mice for bulk RNA-seq (Fig. [82]3A). The sequencing results revealed a total of 1,539 significantly differentially expressed genes (DEGs), with 930 upregulated genes and 609 downregulated genes (Fig. [83]3B–C). Gene set enrichment analysis showed abnormal activation of the Wnt signaling pathway in the oviductal epithelium of the miR-dKO mice. To further evaluate whether other ciliogenesis-related pathways were affected, we analyzed Notch and Hedgehog signaling pathways in the RNA-seq dataset. Comparative pathway enrichment analysis showed that, in contrast to the significant activation of Wnt signaling, neither the Notch nor the Hedgehog pathway displayed substantial enrichment (Fig. [84]3D). These results indicate that Wnt pathway activation is the predominant transcriptional change associated with miR-34/449 deletion. Furthermore, we selected activation-related and silencing-related genes within the Wnt/β-catenin signaling pathway from the DEGs for validation. The results indicated significant upregulation of Wnt pathway activation molecules and downregulation of silencing molecules in both the whole and distal regions of the oviductal epithelium of the miR-dKO mice (Fig. [85]3E, Fig. [86]S4A), while no significant differences were observed in the proximal regions (Fig. [87]S4B). Western blotting revealed a significant increase in β-catenin protein levels in the oviductal epithelium of the miR-dKO mice (Fig. [88]3G). To investigate Wnt/β-catenin pathway activity, we employed the Wnt pathway reporter system, TOPflash/FOPflash assay (Fig. [89]S4C). Primary epithelial cell cultures derived from the oviduct were confirmed to have a purity exceeding 85% of epithelial cells, meeting experimental requirements (Fig. [90]S4D). We conducted the transfection of TOPflash/FOPflash reporter plasmids to the epithelial cells and observed significant activation of the Wnt/β-catenin signaling pathway in the whole and distal regions of the oviducts of the miR-dKO mice, in comparison with those of the control mice (Fig. [91]3H, Fig. [92]S4E), while no significant differences were noted in the proximal regions (Fig. [93]S4F). In conclusion, our findings indicate abnormal activation of the Wnt/β-catenin signaling pathway in the oviductal epithelium of the miR-dKO mice. Fig. 3. [94]Fig. 3 [95]Open in a new tab Wnt/β-catenin signaling pathway hyperactivation in the oviductal epithelium of the miR-dKO mice. A Schematic diagram of transcriptome sequencing (RNA-seq) of mouse oviductal epithelium. B Volcano diagram of differentially expressed genes. Red: 930 upregulated genes; Green: 609 downregulated genes; Blue: 23,292 genes with no significant difference in expression. C Heatmap diagram of RNA-seq. Colors from blue to red indicate the low to high expression of different genes. D Gene set enrichment analysis (GESA) analysis of the Wnt/β-catenin, Notch and Hedgehog signaling pathways. E Relative mRNA expression levels of the genes encoding active and inhibiting molecules of the Wnt signaling pathway in the epithelium of the distal region of the oviduct in the control and the miR-dKO mice. F Venn diagram of upregulated genes of Wnt/β-catenin signaling pathway in differentially expressed genes (DEGs) (yellow) and the predicted target genes of miR-34/449 by Targetscan (purple) and miRDB (blue). G Representative western blot images and statistical results of β-catenin protein levels in the oviduct of the CON and the dKO mice. GAPDH was used as a loading control. H Analysis of the activity of Wnt/β-catenin signaling pathway in the epithelium of the distal region of the oviduct using the TOPflash/FOPFlash assay. I Sequence alignment of the miR-34/449 seed sequence and the binding site in the 3’ untranslated region (3’UTR) of Dvl2 mRNA (red). WT: wild-type sequence of the 3’UTR of Dvl2 mRNA. Mut: mutant sequence of the 3’UTR of Dvl2 mRNA (mutant sites present in green). J The dual-luciferase reporter assay verified that Dvl2 is a target gene of miR-34/449. All the data are presented as the mean ± SD, n = 3. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001 Furthermore, to explore downstream target genes regulated by miR-34/449, we performed a Venn analysis on the predicted target genes from Targetscan and miRDB and the DEGs within the Wnt/β-catenin signaling pathway and identified three differentially expressed target genes (Dvl2, Lef1, Tbl1xr1) (Fig. [96]3F). Dvl2 is a crucial signaling molecule in the Wnt signaling pathway [[97]22–[98]24], which attracted our attention. To confirm Dvl2 as a direct target of miR-34/449, we conducted a dual-luciferase reporter assay by co-transfecting 293 T cells with miR-34/449 mimics and a luciferase reporter construct harboring the Dvl2 3′UTR. This resulted in a modest yet statistically significant reduction in luciferase activity (Fig. [99]3I–J). To validate successful delivery of miR-34/449 mimics and confirm downstream gene repression, we measured miR-34/449 expression levels post-transfection, which showed an ~ tenfold increase compared to the control. Concurrently, endogenous DVL2 protein levels were reduced to ~ 40% of control levels, supporting functional targeting of Dvl2 by miR-34/449 (Fig. [100]S4G–H). Wnt/β-catenin signaling pathway inhibitors rescue the ciliary differentiation defects in the oviductal epithelial organoid derived from the miR-dKO mice To further validate the hypothesis that miR-34/449 regulates the differentiation of ciliated cells through Wnt pathway signaling by targeting Dvl2, we established organoid cultures derived from both control and miR-dKO oviductal epithelia using the BET (B27, EGF, TGF-β receptor kinase inhibitor) growth medium. Compared with the organoids cultured from the control oviductal epithelium, the organoids derived from the miR-dKO oviductal epithelium exhibited faster growth, larger diameters, and greater numbers (Fig. [101]S5A–C). During the organoid culture, epithelial invaginations resembling the folds of the oviductal epithelium in vivo were observed (Fig. [102]S5D), and the organoids could be passaged for extended periods (Fig. [103]S5E). Organoids derived from the miR-dKO mice showed a significant reduction in the number and proportion of Ac-α-tubulin^+ ciliated cells (Fig. [104]S5F). The mRNA (Fig. [105]S5G) and protein-level (Fig. [106]S5H–I) validation also revealed significantly decreased expression of Foxj1 and increased expression of β-catenin and Pax8 in the organoids derived from the miR-dKO mice. In addition, the relative TOP/FOP luciferase activity in the organoids from the miR-dKO mice was higher than that of the control mice (Fig. [107]S5J). Therefore, organoids cultured in the BET medium effectively recapitulate the in vivo oviductal phenotype observed in the miR-dKO mice. Through RNA-seq analysis, we found that, compared with the WT mice, the miR-dKO mice exhibited abnormal activation of the Wnt/β-catenin signaling pathway in the oviductal epithelium. Therefore, we tested whether the addition of Wnt/β-catenin signaling pathway inhibitors, XAV-939 and NSC668036, to the organoid culture system could rescue the ciliated cell differentiation defects observed in the miR-dKO mice (Fig. [108]4A). Using the TOPflash/FOPflash assay, we confirmed that the inhibitors successfully reduced the Wnt signaling pathway activity in the organoids derived from the miR-dKO mice (Fig. [109]4J). Upon continuous observation of the cultured organoids, we found that after treatment with the Wnt pathway inhibitors, the growth rate of the miR-dKO oviductal epithelial organoids significantly slowed down—their size decreased, and their numbers reduced—which tended to be consistent with the growth pattern of the organoids derived from the oviductal epithelia of the control mice (Fig. [110]4B–F). Fig. 4. [111]Fig. 4 [112]Open in a new tab Wnt/β-catenin inhibitors rescue the ciliary differentiation defects in the miR-dKO oviductal epithelial organoids. A Schematic diagram of the oviductal epithelial organoids culture. B Representative images of the culture of oviductal epithelial organoids derived from the miR-dKO mice with or without Wnt signaling pathway inhibitors (XAV-939, NSC668036) and those derived from control (CON) mice. Scale bars: 200 μm. The number and size of the organoids were counted/measured on culture day 4 (B), day 8 (D), and day 16 (E). F The total number of organoids was counted on culture days 8 and 16. G Representative images of co-immunofluorescent staining of Ac-α-tubulin (indicating ciliated cells) and PAX8 (indicating secretory cells) on organoid sections of the control (CON) mice and the miR-dKO mice treated with the Wnt signaling pathway inhibitors or not for 2 months. Scale bars: 50 μm. The box in the lower column shows the higher magnification of the box area in the picture. H Quantification of Ac-α-tubulin^+ cells in the oviductal epithelial cells of the organoids. I Relative mRNA expression levels of β-catenin, Foxj1, Pax8, Dvl2, Cdk1, and Cyclin D1. J The activity of the Wnt signaling pathway in the oviductal epithelial organoids before and after treatment with the Wnt/β-catenin signaling pathway inhibitor was detected by the TOPflash/FOPflash assay. Note that the blue columns in the figure represent the CON group, the purple columns represent the dKO group, the pink columns represent the dKO + XAV939 group and the green columns represent the dKO + NSC668036 group. All the data are presented as the mean ± SD, n = 3. *: p < 0.05; **: p < 0.01 Next, we recovered the cultured organoids and performed immunofluorescence analysis. The results showed that after treatment with the Wnt pathway inhibitors, the number and proportion of Ac-α-tubulin^+ ciliated cells in the miR-dKO oviductal epithelial organoids significantly increased (Fig. [113]4G–H), exhibiting no significant differences compared with those in the control oviductal epithelial organoids. Additionally, it was revealed that after inhibitor treatment, the expression level of Foxj1 mRNA significantly increased, while the mRNA levels of β-catenin, Pax8, Dvl2, Cdk1, and Cyclin D1 significantly decreased in the miR-dKO oviductal epithelial organoids (Fig. [114]4I). Taken together, the above results suggest that Wnt pathway inhibitors can rescue the ciliary differentiation defects in the oviductal epithelial organoids derived from the miR-dKO mice. The miR-34/449 clusters regulate the differentiation of ciliated cells in the oviductal epithelial organoids via the Wnt/β-catenin signaling pathway by targeting Dvl2 Previously, we confirmed that Dvl2, a critical molecule in the Wnt signaling pathway, is a target of miR-34/449 (Fig. [115]3I–J). Moreover, its expression was significantly upregulated in the oviductal epithelium of the miR-dKO mice (Fig. [116]3E). Therefore, we hypothesized that miR-34/449 might influence the activity of the Wnt/β-catenin signaling pathway by targeting Dvl2, thereby regulating the differentiation of ciliated cells. To investigate the relationship between Dvl2 expression and ciliated cell differentiation in the oviductal epithelial organoids of the miR-dKO mice, we used Dvl2-shRNA lentivirus to knock down the expression of Dvl2 in the primary epithelial cells. The infection efficiency of Dvl2-shRNA lentivirus was verified (Fig. [117]S6A–C). Cells positive for infection were selected for organoid culture using the BET culture medium containing 2 mg/ml puromycin (Fig. [118]5A). Continuous observation of the cultured organoids revealed that the organoids derived from the oviductal epithelia of the miR-dKO mice after knocking down Dvl2 (dKO + Dvl2-sh) exhibited reduced growth rates, smaller sizes, and fewer numbers (Fig. [119]5B–F), reaching the growth patterns of the organoids derived from the control mice. The number and proportion of the ciliated cells in the dKO + Dvl2-sh groups increased, but they were still lower than those in the control mice (Fig. [120]5G–H). Furthermore, the mRNA expression levels of Foxj1, Pax8, β-catenin, Dvl2, Cdk1, and Cyclin D1 in the dKO + Dvl2-sh groups were reversed significantly but did not return to those in the organoids derived from the control mice respectively (Fig. [121]5I). The above results suggest that the differentiation of ciliated cells in the miR-dKO oviductal epithelial organoids was partially restored after knocking down Dvl2. Using the TOPflash/FOPflash assay, we verified that the activity of the Wnt/β-catenin signaling pathway significantly decreased in the dKO + Dvl2-sh groups compared with that in the miR-dKO group (Fig. [122]5J). The above results suggest that miR-34/449 likely regulates the activity of the Wnt/β-catenin signaling pathway by targeting Dvl2, thereby influencing the differentiation of ciliated cells. Fig. 5. [123]Fig. 5 [124]Open in a new tab Knockdown Dvl2 partially rescue the ciliary differentiation defects in the miR-dKO oviductal epithelial organoids. A Schematic diagram of the oviductal epithelial organoids culture. B Representative images of the culture of oviductal epithelial organoids derived from the miR-dKO mice treated with or without Dvl2-shRNA1 (Dvl2-sh1) or Dvl2-shRNA2 (Dvl2-sh2) lentivirus and those derived from control (CON) mice. Scale bars: 200 μm. The number and size of the organoids were counted/measured on culture day 4 (C), day 8 (D), and day 16 (E). F The total number of organoids was counted on culture day 8 and day 16. G Representative images of co-immunofluorescent staining of Ac-α-tubulin (indicating ciliated cells) and PAX8 (indicating secretory cells) on organoid sections of the CON mice and the miR-dKO mice treated with Dvl2-sh1 or Dvl2-sh2 lentivirus or not. Scale bars: 50 μm. The box in the lower column shows the higher magnification of the box area in the picture. H Quantification of Ac-α-tubulin^+ cells in the oviductal epithelial organoids. I Relative mRNA expression levels of β-catenin, Foxj1, Pax8, Dvl2, Cdk1, and Cyclin D1 in different organoids. J The activity of the Wnt/β-catenin signaling pathway in oviductal epithelial organoids with or without treatment of Dvl2-sh1 or Dvl2-sh2 lentivirus detected by the TOPflash/FOPflash assay. Note that the blue columns in the figure represent the CON group, the purple columns represent the dKO group, the pink columns represent the dKO + Dvl2-sh1 group and the green columns represent the dKO + Dvl2-sh2 group. All the data are presented as the mean ± SD, n = 3. *: p < 0.05; **: p < 0.01 In addition, we utilized Dvl2-overexpression (Dvl2-OE) lentiviral vectors with or without miR-34/449 mimics to infect organoids derived from the control mice (Fig. [125]6A), verifying the function of miR-34/449 in regulating oviductal ciliated cell differentiation by targeting Dvl2. The infection efficiency of Dvl2-OE lentivirus was verified (Fig. [126]S7A–C). Infected cells were selected for organoid cultivation. Continuous observation of the cultivated organoids revealed that organoids derived from the control mice after overexpression of Dvl2 (CON + Dvl2-OE) phenocopied the organoids derived from the miR-dKO mice. However, the phenotype was reversed after treatment with miR-34/449 mimics (Fig. [127]6B–F). Overexpression of Dvl2 in the control organoids alone also induced a significant decrease in the number of ciliated cells, but it could be reversed after the addition of miR-34/449 mimics (Fig. [128]6G–H). Interestingly, we found that the expression levels of Cdk1 and Cyclin D1, two cell cycle regulating proteins, increased with the overexpression of Dvl2 in the control organoids, similar to the expression levels in the miR-dKO organoids, which could be reversed after treating with miR-34/449 mimics (Fig. [129]6I). Furthermore, we found that the Wnt/β-catenin signaling activity significantly increased in the control organoids after Dvl2-overexpression and could be reversed after treating with miR-34/449 mimics (Fig. [130]6J). Taken together, these results collectively suggest that Dvl2 overexpression alone in the control oviductal epithelial organoids can elevate Wnt/β-catenin signaling activity, leading to differentiation impairment in ciliated cells through being targeted by miR-34/449. Fig. 6. [131]Fig. 6 [132]Open in a new tab Dvl2 overexpression impairs ciliated cell differentiation in the control oviductal epithelial organoids. A Schematic diagram of the oviductal epithelial organoids culture. B Representative images of the culture of oviductal epithelial organoids derived from the control (CON) mice treated with or without miR-34/449 mimics following treatment with Dvl2 overexpression (Dvl2-OE) and those derived from the miR-dKO mice. Scale bars: 200 μm. The number and sizesof the organoids were counted/measured on culture day 4 (C), day 8 (D), and day 16 (E). F The total number of organoids was counted on culture day 8 and day 16. G Representative images of co-immunofluorescent staining of Ac-α-tubulin (indicating ciliated cells) and PAX8 (indicating secretory cells) on organoid sections of the miR-dKO and the control mice treated with or without Dvl2-OE and miR-34/449 mimics. Scale bars: 50 μm. The box in the lower column shows the higher magnification of the box area in the picture. H Quantification of Ac-α-tubulin^+ cells in oviductal epithelial organoids. I Relative mRNA expression levels of β-catenin, Foxj1, Pax8, Dvl2, Cdk1, and CyclinD1 in different organoids. J The activity of the Wnt/β-catenin signaling pathway in the oviductal epithelial organoids with or without Dvl2-OE and miR-34/449 mimics was detected by the TOPflash/FOPflash assay. Note that the blue columns in the figure represent the CON group, the purple columns represent the dKO group, the pink columns represent the CON + Dvl2-OE group, and the green columns represent the CON + Dvl2-OE + mimics group. All the data are presented as the mean ± SD, n = 3. *: p < 0.05; **: p < 0.01 Discussion In the present study, we have found that miR-34/449 regulates the Wnt/β-catenin signaling pathway by targeting Dvl2 to affect oviductal ciliated cell differentiation and oviductal epithelial homeostasis and ultimately affect fertility in mice. In the context of the oviduct, the homeostasis of epithelial cells is particularly important, as the oviduct is a vital site for oocyte pickup, gamete and embryo transport, fertilization, and early embryonic development [[133]6]. The epithelial cells, including ciliated cells and secretory cells, work in concert to support these processes. We found that ablation of miR-34b/c and miR-449 leads to the disrupted differentiation of ciliated cells only in the distal part of miR-dKO mice, which led to a decrease in the number of ciliated cells and ultimately resulted in oocyte pickup disorder and infertility. The result is consistent with a previous report, which demonstrates that motile cilia in the infundibulum of the oviduct are essential for oocyte pickup and thus, female fertility, whereas motile cilia in other parts of the oviduct facilitate gamete and embryo transport but are not required for female fertility [[134]17]. In addition, we also found that the differentiation of ciliated cells in the proximal regions of the oviduct in the miR-dKO mice showed no significant differences. Regional differences in the impact of miR-34/449 deletion on epithelial differentiation are likely multifactorial. One possibility is that progenitor cells in the distal regions of the oviduct are intrinsically more dependent on precise Wnt/β-catenin signaling regulation to establish and maintain ciliated cell identity, whereas proximal regions may exhibit reduced sensitivity to Wnt pathway perturbation due to differences in baseline signaling thresholds [[135]25, [136]26]. This spatial distribution suggests that distal epithelial progenitors rely more heavily on miR-34/449-mediated regulation to suppress Wnt/β-catenin signaling during the transition toward ciliated cell fate. In contrast, the proximal oviductal epithelium may depend on alternative regulatory pathways, such as Notch or retinoic acid signaling, or exhibit lower baseline Wnt activity and differential inflammatory signaling, as has been reported in the human fallopian tube [[137]27, [138]28]. These differences may underlie the selective susceptibility of the distal epithelium to miR-34/449 loss of function. Further investigations using spatial transcriptomics and functional assays will be required to clarify how these compartmentalized regulatory mechanisms contribute to epithelial heterogeneity along the oviduct. It has been previously reported that to sustain the stemness of the oviductal epithelial cells during the cultivation of oviductal epithelial organoids, a diverse array of activators of the Wnt signaling pathway, including Wnt3a and Rspondin are added in the culture medium [[139]19]. However, this was not suitable for testing the mechanism of ciliated cell differentiation in our study. Building upon the pioneering work by Ying Xie and colleagues [[140]20], we progressively streamlined the composition of the culture medium, culminating in the formulation known as the BET medium (B27, EFG, TGF-β receptor kinase inhibitor), and subsequently utilized the BET medium in the cultivation of oviductal epithelial organoids to validate the mechanism underpinning the differentiation of oviductal epithelial ciliated cells in our research. We discovered the hyperactivation of the Wnt/β-catenin signaling pathway within the oviducts of miR-dKO mice. Subsequently, in the culture system of oviductal epithelial organoids derived from the miR-dKO mice, we introduced two Wnt/β-catenin signaling pathway inhibitors, XAV-939 and NSC668036, to evaluate whether curtailing the excessive activation of the Wnt pathway could ameliorate the differentiation impediment observed in the oviductal ciliated cells in the miR-dKO mice. XAV-939 inhibits the activity of the Wnt/β-catenin signaling pathway by promoting the degradation of β-catenin [[141]29, [142]30]. NSC668036 disrupts the interaction n between DVL and the frizzled receptor by binding to the PDZ domain of DVL and impedes the transduction of the Wnt/β-catenin signal [[143]31, [144]32]. The application of these inhibitors yielded compelling results in our study, as evidenced by a substantial increase in the population of ciliated cells within the oviductal epithelial organoids derived from the miR-dKO mice. Furthermore, the expression levels of pertinent molecular markers were restored to baseline levels. The results substantiated the hypothesis that the differentiation impairment of ciliated cells within the oviductal epithelium of miR-dKO mice is indeed attributable to the aberrant activation of the Wnt/β-catenin signaling pathway and underscored the therapeutic potential of Wnt pathway inhibitors in mitigating this impairment. The Wnt/β-catenin signaling pathway exerts a pivotal influence on cell proliferation and differentiation. Many studies have shown that hyperactivation of the Wnt/β-catenin signaling pathway can lead to excessive cell proliferation and impede cell differentiation [[145]33–[146]37]. Arnab Ghosh and his colleagues have reported that the Wnt/β-catenin signaling pathway is active during postnatal oviductal epithelial maturation and differentiation. Using a mouse model to conditionally over-activate β-catenin in oviductal secretory cells, they found expansion of secretory cells in mutant oviducts [[147]8]. In our study, we demonstrated that Wnt signaling was hyperactivated in the oviduct of miR-dKO mice, which may be the reason for the faster growth and larger size of the oviductal epithelial organoids collecting from the miR-dKO mice. At the same time, because of the abnormal activation of the Wnt signaling pathway in the oviductal epithelium of miR-dKO mice, the differentiation of secretory cells into ciliated cells was impeded, resulting in the reduction in ciliated cells. Dvl2 is a key molecule of the Wnt signaling pathway targeted by miR-34/449, which was overexpressed in the oviductal epithelium of the miR-dKO mice. We found that downregulation of Dvl2 in the oviductal epithelial organoids derived from the miR-dKO mice led to partial restoration of ciliated cell differentiation while using Wnt signaling pathway inhibitors could fully rescue the aberrant differentiation of ciliated cells. The reasons for the discrepancy may include: 1. The knockdown efficiency of Dvl2-shRNA. Although the expression of Dvl2 was reduced after Dvl2-shRNA lentiviral treatment in the miR-dKO mice, it remained higher than that of control mice (Fig. [148]5I). This resulted in slightly higher activity of the Wnt/β-catenin signaling pathway, in comparison with that of the control mice (Fig. [149]5J), thereby partially rescuing the ciliated cell differentiation. 2. Complex relationships between miRNA and target genes. The relationships between miRNAs and target genes are intricate, whereby one miRNA can target multiple mRNAs, and conversely, one mRNA can be regulated by multiple miRNAs [[150]38, [151]39]. We performed target gene prediction of miR-34/449 based on the upregulated genes in the Wnt signaling pathway in the oviductal epithelia of miR-dKO mice, and three possible target genes—Dvl2, Lef1, and Tbl1xr1—were obtained. We chose to study Dvl2 because its expression was increased more significantly, and it is a key transmitter in the Wnt signaling pathway. The synergistic effects of the other two target genes may also influence the activity of the Wnt/β-catenin signaling pathway [[152]40, [153]41], which need to be further investigated. Although it is known that deletion of highly expressed miRNAs can, in some contexts, lead to compensatory upregulation of other miRNAs due to reduced competition for components of the miRNA processing machinery [[154]38], several lines of evidence in our study support the conclusion that the observed phenotypes are primarily attributable to the loss of miR-34/449 clusters. Specifically, the robust functional rescue achieved by pharmacological inhibition of Wnt/β-catenin signaling and by targeted Dvl2 knockdown strongly indicates that derepression of this pathway is a key driver of impaired ciliated cell differentiation. Furthermore, the unique targeting properties of miR-34/449 family members and the consistency of our findings with previously reported phenotypes in other multiciliated tissues [[155]14, [156]15] argue against significant compensation by unrelated miRNAs. Nevertheless, we acknowledge that broader changes in the miRNA landscape cannot be entirely excluded and may contribute to the phenotype to a limited extent. Future studies employing comprehensive small RNA profiling may further elucidate potential compensatory mechanisms. Conclusions In this study, by using both the miR-34/449-dKO mouse model and the oviductal epithelial organoid model, the results presented here demonstrate that miR-34/449 are instrumental in regulating the activity of the Wnt/β-catenin signaling pathway by targeting Dvl2, thereby exerting a profound influence on the differentiation of ciliated cells within the oviductal epithelium. It would be interesting to test whether this new mechanism of ciliated cell differentiation in the oviduct exists in other multiciliated organs. Fundings This work was supported by the Shanghai Natural Science Foundation Grant (20ZR1430700 to J.W.), the National Natural Science Foundation of China (81671509 to J. W.) and the Shanghai Municipal Health Commission Foundation Grant (202340286 to J.W.). Supplementary Information Below is the link to the electronic supplementary material. [157]Supplementary Material 1^ (377.8KB, docx) [158]Supplementary Material 2^ (12.4MB, docx) Acknowledgements