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

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