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Primary cilia are shear stress calcium-responsive mechanosensors in the epididymis

ABSTRACT: The epithelium of the epididymis proliferates and differentiates until puberty, to become fully functional when the first wave of spermatozoa initiates its post-testicular maturation process. While the tension exerted by the testicular-derived fluid has been proposed to stimulate the proliferation/differentiation of the epithelium through physical cues, the mechanism involved has never been identified. Here we assessed whether the responsiveness of the epididymal cells to luminal shear stress depends on functional primary cilia. Combining in silico fluidic modeling with in vitro microfluidic strategies, we subjected immortalized distal caput epididymis principal cells (DC2) to a static or fluid shear stress condition. RNA sequencing studies identified 100 genes as being overexpressed in response to shear stress (Fold change >1.5, FDR< 0.05), including Early growth response (EGR) 2/3, Cellular communication network factor (CCN) 1/2, and Fos, also found induced in flow-stimulated renal cells. Enrichment of transcriptional factors involved in proliferative signaling pathways was consistent with the proliferative changes observed in DC2 cells under shear stress conditions. Of interest, DEGs particularly relevant to epididymal physiology and sperm maturation, including Serpine1 and Adamts1, were identified as secreted factors interacting with the maturing spermatozoa. While shear stress triggered a rapid increase of intracellular Ca2+ in DC2 cells, this response was abrogated following the impairment of primary ciliogenesis through pharmacological and siRNA approaches. Embracing physical and biological concepts, our findings identify primary cilia as a fluid-mechanosensor that may contribute to epididymal cell development and proliferation, a requisite to sperm maturation

ORGANISM(S): Mus musculus

PROVIDER: GSE269872 | GEO | 2025/01/22

REPOSITORIES: GEO

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Project description:Background. Primary cilia (PC) are solitary antennae present at the cell surface. These non-motile cilia play an important role in organ development and tissue homeostasis through the transduction of the Hedgehog (Hh) signaling pathway. We recently revealed the presence of PC in the epithelium of the developing epididymis, an organ of the male reproductive system whose dysfunction triggers male infertility. Acknowledging that systemic blockade of the Hh pathway trigger epididymal dysfunctions in vivo, our main goals were 1) to portray the epididymal Hh environment, 2) to determine the direct responsiveness of epididymal epithelial cells to Hh, and 3) to define the contribution of PC to the transduction of this pathway. Results. The Hh ligands Indian and Sonic hedgehog (Ihh and Shh) were respectively located in principal and clear cells of the mouse epididymis by immunofluorescent staining. The propensity of epididymal principal cells to respond to Hh signaling was assessed on immortalized epididymal DC2 cells by western-blot, confocal imaging and 3D-reconstruction. Our results indicate that epididymal principal cells secrete Ihh and expose PC that co-localize with the conventional acetylated tubulin/Arl13b ciliary markers, as well as with GLI3 Hh signaling factor. Gene expression microarray profiling indicated that the expression of 43 and 248 genes was respectively and significantly modified following pharmacological treatment of DC2 cells with the Hh agonist SAG (250 nM) or the Hh antagonist cyclopamine (20 µM) compared with the control. Among Hh target genes identified, 6.7 % presented perfect matches for GLI-transcription factor consensus sequences, and the majority belonged to interferon-dependent immune response and lipocalin 2 pathways. Finally, the contribution of epididymal PC to the transduction of canonical Hh pathway was validated by ciliobrevinD treatment, which induced a significant decrease of PC length and the expressional reduction of Hh signalling targets. Conclusions. All together our data indicate that PC from epithelial principal cells regulate gene expression profile through a possible autocrine Hh signaling. This provides new hypotheses regarding the potential contribution of PC and Hh signaling in intercellular cross-talk and immunological regulation of the epididymis.

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Primary cilia are shear stress calcium-responsive mechanosensors in the epididymis

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