title: Primary cilia on porcine testicular somatic cells and their role in hedgehog signaling and tubular morphogenesis in vitro.
abstract: The primary cilium is a microtubule-based sensory organelle found on nearly all eukaryotic cells but little is understood about its function in the testis. We investigate the role of primary cilia on testis cells in vitro by inhibiting formation of the primary cilium with Ciliobrevin D, a cell-permeable, reversible chemical inhibitor of ATPase motor cytoplasmic dynein. We analyzed cultured cells for the presence of primary cilia and their involvement in hedgehog signaling. Primary cilia were present on 89.3 ± 2.3 % of untreated testicular somatic cells compared to 3.1 ± 2.5 % cells with primary cilia for Ciliobrevin D-treated cells. Protein levels of Gli-2 and Smoothened were lower on Western blots after suppression of cilia with Ciliobrevin D. The inhibitor did not affect centrosome localization or cell proliferation, indicating that changes were due to ablation of the primary cilium. Testicular somatic cells have the ability to form three-dimensional tubules in vitro. In vitro-formed tubules were significantly longer and wider in the control group than in the Ciliobrevin D-treated group (9.91 ± 0.35 vs. 5.540 ± 1.08 mm and 339.8 ± 55.78 vs. 127.2 ± 11.9 μm, respectively) indicating that primary cilia play a role in tubule formation. Our results establish that the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, affects the hedgehog signaling pathway and impairs tubule formation in vitro. These findings provide evidence for a role of cilia in the testis in cell signaling and tubular morphogenesis in vitro.
authors.affiliation: Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, HMRB 404, Calgary, Alberta, T2N 4N1, Canada.
authors.forename: Camila
authors.initials: C
authors.lastname: Dores
authors.affiliation: Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, HMRB 404, Calgary, Alberta, T2N 4N1, Canada.
authors.forename: Whitney
authors.initials: W
authors.lastname: Alpaugh
authors.affiliation: Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, HMRB 404, Calgary, Alberta, T2N 4N1, Canada.
authors.forename: Lin
authors.initials: L
authors.lastname: Su
authors.affiliation: Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, HMRB 404, Calgary, Alberta, T2N 4N1, Canada.
authors.forename: Jeff
authors.initials: J
authors.lastname: Biernaskie
authors.affiliation: Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, HMRB 404, Calgary, Alberta, T2N 4N1, Canada. idobrins@ucalgary.ca.
authors.forename: Ina
authors.initials: I
authors.lastname: Dobrinski
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism; D002923:Cilia / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D053823:Hedgehog Proteins / Q000378:metabolism*; D008297:Male; D009024:Morphogenesis / Q000187:drug effects; D052999:Quinazolinones / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D034421:Sus scrofa; D013737:Testis / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D053823:Hedgehog Proteins; D052999:Quinazolinones; C000595851:ciliobrevin D
keywords: Hedgehog; Primary cilia; Somatic cells; Testis; Tubular morphogenesis