Dicer1 is an endoribonuclease involved in the biogenesis of functional molecules such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs). These small non-coding RNAs are important regulators of post-transcriptional gene expression and participate in the control of male fertility. With the knowledge that 1) Dicer1-dependent factors are required for proper sperm maturation in the epididymis, and that 2) miRNAs are potent mediators of intercellular communication in most biological systems, we investigated the role of Dicer1-dependent factors produced by the proximal epididymis (initial segment/caput)- including miRNAs- on the regulation of epididymal gene expression in the distal epididymis regions (i.e. corpus and cauda). To this end, we performed comparative microarray and ANOVA analyses on control vs. Defb41iCre/wt;Dicer1fl/fl mice in which functional Dicer1 is absent from the principal cells of the proximal epididymis. We identified 35 and 33 transcripts that displayed significant expression level changes in the corpus and cauda regions (Fold change > 2 or < −2; p < 0.002), respectively. Among these transcripts, Zn-alpha 2-glycoprotein (Azgp1) encodes for a sperm equatorial protein whose expression in the epididymis of Dicer1 cKO mice is significantly increased compared to controls. In addition, 154 miRNAs, including miR-210, miR-672, miR-191 and miR-204, showed significantly impaired biogenesis in the absence of Dicer1 from the principal cells of the proximal epididymis (Fold change > 2 or < −2; p < 0.01). These miRNAs are secreted via extracellular vesicles (EVs) derived from the DC2 epididymal principal cell line, and their expression correlates with target transcripts involved in distinct biological pathways, as evidenced by in silico analysis. Albeit correlative and based on in silico approach, our study proposes that Dicer1-dependent factors trigger- directly or not—significant genes expression changes in distinct regions of this organ. The paracrine control of functions important to post-testicular sperm maturation by Dicer1-dependent factors may open new avenues for the identification of molecular targets important to male fertility control.

Study funded by an NSERC operating grant to CB (RGPIN-2015-109194). No competing interest to declare.

30Primary cilia are sensory organelles that orchestrate major signaling pathways during organ 31 development and homeostasis. By using a double Arl13b/mCherry-Cetn2/GFP transgenic mouse 32 model, we characterized the spatio-temporal localization of primary cilia in the epididymis, from 33 birth to adulthood. We report here a constitutive localisation of primary cilia in peritubular 34 myoid cells and a dynamic profiling in differentiated epithelial cells throughout post-natal 35 development. While primary cilia are present at the apical pole of the undifferentiated epithelial 36 cells from birth to puberty, they are absent from the apical pole of the epithelium in adults, 37where they appear exclusively associated with cytokeratin 5-positive basal cells. Exogenous 38 labeling of primary cilia marker Arl13b and IFT88 confirmed the cell lineage specific localization 39 of primary cilia in basal cells and myoid cells in human epididymides. From whole epididymis 40 tissues and serum-free cultures of DC2 murine epididymal principal cell lines we determined 41 that primary cilia from the epididymis are associated with the polycystic kidney disease-related 42 proteins polycystin 1 (PC1) and polycystin 2 (PC2), and Gli3 Hedgehog signaling transcription 43 factor. Thus, our findings unveil the existence of primary cilia sensory organelles, which have the 44 potential to mediate mechano/ chemo-signaling events in the epididymis. 45