Journal: Biology of Reproduction

Article Title: Induction of in vivo -like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells ^†

doi: 10.1093/biolre/ioae090

Figure Lengend Snippet: (a) Representative images of re-differentiated equine oviduct epithelial cell (REOEC) monolayers cultured for 1 month in (A) 0%, (B) 30%, (C) 50%, (D) 70% and (E) 100% Wnt3a containing medium or for 7 days in (F) 0 μM, (G) 1 μM, (H) 3 μM, (I) 5 μM, and (J) 10 μM Wnt agonist CHIR 99021. No effect on cell or nucleus morphology was observed after 1 month culture in 30% Wnt3a containing medium. Epithelial-mesenchymal transition was observed within 1 month in re-differentiated EOEC monolayers cultured in 50 and 70% Wnt3a containing medium, with cells becoming elongated with enlarged round to oval nuclei, growing in multiple cell layers, and lacking primary cilia. A similar effect was observed after 1 week exposure to 1, 3, and 5 μM Wnt agonist CHIR 99021. The EOECs detached from the polycarbonate membrane in 100% Wnt3a conditioned medium and 10 μM Wnt agonist CHIR 99021. Moreover, none of the tested conditions supported cilia formation [green (anti-acetylated α-tubulin antibodies-AlexaFluor 488 secondary antibody): primary (single green dots at each EOEC) and secondary cilia (many green dots on some EOECs), red (phalloidin conjugated to Alexa Fluor 568): cytoskeleton, blue (Hoechst 33342): nuclei] (original magnification, 400x – scale bar: 25 μm). (b) The effect on transepithelial electrical resistance (TEER) of confluent re-differentiated EOEC monolayers cultured in 0%, 30%, 50%, 70%, and 100% Wnt3a containing medium after 1, 2, 3, and 4 weeks of incubation is shown. At 3 weeks, TEER values dropped below the confluency threshold (indicated by the horizontal line) in 50% and 70% Wnt3a containing medium; a similar observation was observed after 1 week in 100% Wnt3a containing medium ( n = 3 mares; 1 insert per mare per concentration at each time point). Values that differ significantly between TEER values are indicated by small letters. (c) The effect on TEER of confluent re-differentiated EOEC monolayers exposed to CHIR 99021 (Wnt ligand; 0, 1, 3, 5, and 10 μM), IWP-2 (Wnt inhibitor; 0, 2, and 20 μM), JAG-1 (Notch ligand; 0, 1, and 10 μM) and DBZ (Notch inhibitor; 0, 1, and 10 μM) was assessed after 1 week. TEER values dropped below the confluency threshold (indicated by the horizontal line) under 1, 3, 5, and 10 μM CHIR 99021 conditions ( n = 3 mares; 1 insert per mare for each concentration). Values that differ significantly between TEER values are indicated by small letters.

Article Snippet: Next, the monolayers were washed twice in PBS containing 0.5% Triton X-100 for 5 minutes, before overnight incubation at 4°C with a monoclonal mouse anti-acetylated α-tubulin primary antibody (sc-23 950, 1:100 dilution, Santa Cruz Biotechnology, Immunologic, Duiven, the Netherlands).

Techniques: Cell Culture, Membrane, Incubation, Concentration Assay

Journal: Biology of Reproduction

Article Title: Induction of in vivo -like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells ^†

doi: 10.1093/biolre/ioae090

Figure Lengend Snippet: (a) Representative overview image of a spontaneously re-differentiated equine oviduct epithelial cell (REOEC) monolayer 21 days after air-liquid interface introduction [green (anti-acetylated α-tubulin antibodies-AlexaFluor 488 secondary antibody): primary (single green dots at each EOEC) and secondary cilia (many green dots on some EOECs), red (phalloidin conjugated to Alexa Fluor 568): cytoskeleton, blue (Hoechst 33342): nuclei] (original magnification, 100x – scale bar: 100 μm). (b) Two images of the same area of a REOEC monolayer, showing that (A) secondary cilia were present mainly in spontaneously REOEC monolayer areas with (B) a higher cell density (left of white line) (original magnification, 400x – scale bar: 25 μm). (b) The effect of varying seeding density of trypsinized de-differentiated EOECs from 0.1 to 10 × 10 5 cells per 0.33 cm 2 insert membrane was assessed 1 month after air-liquid interface introduction. No relationship was observed between EOEC seeding density and secondary ciliation rates in spontaneously re-differentiated EOEC monolayers. Data are mean (± SD) percentage of secondary ciliated cells ( n = 3 mares; 3 inserts per mare per seeding concentration).

Article Snippet: Next, the monolayers were washed twice in PBS containing 0.5% Triton X-100 for 5 minutes, before overnight incubation at 4°C with a monoclonal mouse anti-acetylated α-tubulin primary antibody (sc-23 950, 1:100 dilution, Santa Cruz Biotechnology, Immunologic, Duiven, the Netherlands).

Techniques: Membrane, Concentration Assay

Journal: Biology of Reproduction

Article Title: Induction of in vivo -like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells ^†

doi: 10.1093/biolre/ioae090

Figure Lengend Snippet: Representative (a) bright field (differential interference contrast) light images of de-differentiated equine oviduct epithelial cell (EOEC) monolayers 10 days after oviduct explant seeding in conventional wells (magnification 100x – scale bar: 100 μm) and (b) fluorescent images of diffusely ciliated re-differentiated EOEC monolayers 1 month after air-liquid interface introduction in hanging inserts. If (A) 1 × 10 6 or (B) 5 × 10 6 oviduct explant cells were initially seeded in conventional wells to obtain de-differentiated EOEC monolayers, diffuse ciliated EOEC monolayers were obtained after re-differentiation. In contrast, seeding (C) 10 × 10 6 or (D) 30 × 10 6 oviduct explant cells resulted in monolayers with <2% ciliated EOEC after re-differentiation [green (anti-acetylated α-tubulin antibodies-AlexaFluor 488 secondary antibody): primary (single green dots at each EOEC) and secondary cilia (many green dots on some EOECs), red (phalloidin conjugated to Alexa Fluor 568): cytoskeleton, blue (Hoechst 33342): nuclei] (original magnification, 400x – scale bar: 25 μm). (c) The effect on secondary cilia formation of varying oviduct explant cell seeding concentration from 1 × 10 6 to 30 × 10 6 cells per 9.6 cm 2 well to establish de-differentiated EOEC monolayers, prior to re-differentiation with an air-liquid interface for 1 month. For comparison, in vivo ±60% of EOECs exhibit secondary cilia. Using a lower oviduct explant cell concentration to obtain de-differentiated EOEC monolayers was critical to obtaining spontaneously re-differentiated EOEC monolayers with secondary cilia in a diffuse pattern. Data are mean (± SD) percentages of secondary ciliated cells ( n = 3 mares; 3 inserts per mare per seeding concentration). Values that differ significantly between EOEC concentration are indicated by small letters.

Article Snippet: Next, the monolayers were washed twice in PBS containing 0.5% Triton X-100 for 5 minutes, before overnight incubation at 4°C with a monoclonal mouse anti-acetylated α-tubulin primary antibody (sc-23 950, 1:100 dilution, Santa Cruz Biotechnology, Immunologic, Duiven, the Netherlands).

Techniques: Concentration Assay, Comparison, In Vivo

Journal: Biology of Reproduction

Article Title: Induction of in vivo -like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells ^†

doi: 10.1093/biolre/ioae090

Figure Lengend Snippet: (a) The combined effect of oviduct explant cell seeding concentration and Notch inhibition on secondary cilia formation. De-differentiated equine oviduct epithelial cells (EOECs) were obtained 10 days after seeding oviduct explant cells at concentrations varying from 1 to 30 × 10 6 cells per 9.6 cm 2 well. Subsequently, secondary ciliation rates in re-differentiated EOEC monolayers were assessed 1 month after air-liquid interface introduction. During the last week of culture, EOEC monolayers were incubated in the absence or presence of 10 μM DBZ. A lower oviduct explant cell concentration to obtain de-differentiated EOEC monolayers supported a diffuse ciliation pattern after re-differentiation. Subsequent exposure to DBZ enhanced ciliation rates up to a patchy ciliation pattern showing ciliation rates similar to the in vivo situation (± 60% secondary ciliated EOECs; ). In contrast, initially seeding 10 or 30 × 10 6 oviduct explant cells resulted in <2% secondary ciliated EOECs after re-differentiation, irrespective of DBZ. Data are mean (± SD) percentage of secondary ciliated EOECs ( n = 3 mares; 3 inserts per mare per seeding concentration). Values that differ between oviduct explant cell concentration are indicated by small letters. (b) Representative fluorescent images of re-differentiated EOEC monolayers 1 month after air-liquid interface introduction. To obtain (A) diffuse ciliated EOEC monolayers, a maximum seeding concentration of 5 × 10 6 oviduct explant cells was required. Subsequent exposure to DBZ during re-differentiation supported the development of (C) in vivo -like patchy ciliated EOEC monolayers. (B, D), Higher oviduct explant cell seeding concentration did not support secondary ciliation, regardless of whether re-differentiated EOEC monolayers were exposed to DBZ [green (anti-acetylated α-tubulin antibodies-AlexaFluor 488 secondary antibody): primary (single green dots at each EOEC) and secondary cilia (many green dots on some EOECs), red (phalloidin conjugated to Alexa Fluor 568): cytoskeleton, blue (Hoechst 33342): nuclei] (original magnification, 400x – scale bar: 25 μm).

Article Snippet: Next, the monolayers were washed twice in PBS containing 0.5% Triton X-100 for 5 minutes, before overnight incubation at 4°C with a monoclonal mouse anti-acetylated α-tubulin primary antibody (sc-23 950, 1:100 dilution, Santa Cruz Biotechnology, Immunologic, Duiven, the Netherlands).

Techniques: Concentration Assay, Inhibition, Incubation, In Vivo

Journal: Biology of Reproduction

Article Title: Induction of in vivo -like ciliation in confluent monolayers of re-differentiated equine oviduct epithelial cells ^†

doi: 10.1093/biolre/ioae090

Figure Lengend Snippet: (a) Representative fluorescent images showing the secretory marker clathrin (green) in (A, F) in vivo oviduct tissue sections from mares in the luteal and follicular phase, respectively, and in re-differentiated EOEC monolayers after (B, C, D, E) 14 days of luteal (oviduct concentration: 10 ng/mL E2 and 1000 ng/mL P4) and (G, H, I, J) 7 days of subsequent follicular phase hormone exposure (oviduct concentration: 80 ng/mL E2 and 40 ng/mL P4). Higher clathrin expression was observed for re-differentiated EOEC monolayers exposed to follicular than to luteal phase hormone conditions; [pink (anti-acetylated α-tubulin antibodies-Alexa Fluor 647 secondary antibody): primary (single pink dots at each EOEC) and secondary cilia (many pink dots on some EOECs), green (anti-clathrin antibodies- Alexa Fluor 488 secondary antibody) red (phalloidin conjugated to Alexa Fluor 568): cytoskeleton, blue (Hoechst 33342): nuclei] (original magnification, 400x; A, F - scale bar: 50 μm; B, C, D, E, G, H, I, J – scale bar: 25 μm). (b) Cross sections through re-differentiated EOEC monolayers confirms higher clathrin expression after exposure to (B) follicular compared to (A) luteal phase hormonal conditions. Moreover, clathrin expression was observed in ciliated and non-ciliated EOECs. (C) Effect of estradiol and progesterone to mimic estrous cycle phase on mean clathrin fluorescence intensity per cell in re-differentiating EOECs, after 14 days of luteal and 7 days of subsequent follicular phase hormone exposure. Significantly higher clathrin expression in re-differentiated EOECs was observed after follicular phase hormone exposure. Data are mean (± SD) clathrin fluorescence intensity per cell ( n = 3 mares; 3 inserts per mare per hormonal condition). Values that differ between clathrin fluorescence intensity per cell are indicated by small letters.

Article Snippet: Next, the monolayers were washed twice in PBS containing 0.5% Triton X-100 for 5 minutes, before overnight incubation at 4°C with a monoclonal mouse anti-acetylated α-tubulin primary antibody (sc-23 950, 1:100 dilution, Santa Cruz Biotechnology, Immunologic, Duiven, the Netherlands).

Techniques: Marker, In Vivo, Concentration Assay, Expressing, Fluorescence

Journal: Life Science Alliance

Article Title: CC2D1A causes ciliopathy, intellectual disability, heterotaxy, renal dysplasia, and abnormal CSF flow

doi: 10.26508/lsa.202402708

Figure Lengend Snippet: (A) Western blot of fibroblast cell line lysates from control, and Family#1 father, female patient, and male patient. The affected patient cell lines did not show detectable CC2D1A expression. (B, C) Quantification of fibroblast starvation–induced ciliary length. The patients displayed fewer ciliated cells or shorter ciliary lengths. ns, not significant; P < 0.01 (**), P < 0.0001 (****). (D, E, F) Cultured and starved fibroblasts from Family#1 father, female patient, and male patient stained with DAPI (blue) to show nuclei, phalloidin, Alexa Fluor 488 (green) to show actin cytoskeleton, and anti-acetylated tubulin (red) to show ciliary axonemes. Cilia on the patient fibroblasts were less frequent and shorter than those on the control and father fibroblasts. Scale bar = 25 μm. Source data are available for this figure.

Article Snippet: Cells were then incubated in blocking buffer (3% BSA/PBS with 0.1% Triton X-100) for 1 h, then incubated overnight at 4°C in a primary antibody (mouse anti-acetylated tubulin; Sigma-Aldrich) diluted 1:1,000 in blocking buffer.

Techniques: Western Blot, Control, Expressing, Cell Culture, Staining