Hedgehog signaling regulates Wolffian duct development through the primary cilium†.

Primary cilia play pivotal roles in embryonic patterning and organogenesis through transduction of the Hedgehog signaling pathway (Hh). Although mutations in Hh morphogens impair the development of the gonads and trigger male infertility, the contribution of Hh and primary cilia in the development of male reproductive ductules, including the epididymis, remains unknown. From a Pax2Cre; IFT88fl/fl knock-out mouse model, we found that primary cilia deletion is associated with imbalanced Hh signaling and morphometric changes in the Wolffian duct (WD), the embryonic precursor of the epididymis. Similar effects were observed following pharmacological blockade of primary cilia formation and Hh modulation on WD organotypic cultures. The expression of genes involved in extracellular matrix, mesenchymal-epithelial transition, canonical Hh and WD development was significantly altered after treatments. Altogether, we identified the primary cilia-dependent Hh signaling as a master regulator of genes involved in WD development. This provides new insights regarding the etiology of sexual differentiation and male infertility issues.

Hedgehog signaling pathway regulates gene expression profile of epididymal principal cells through the primary cilium.

Primary cilia (PC) are organelles that sense and respond to dynamic changes of the extracellular milieu through the regulation of target genes. By using the epididymis as a model system, we determined the contribution of primary cilia in the regulation of epithelial cell functions through the transduction of the Hedgehog (Hh) signaling pathway. Both Sonic (SHH) and Indian Hedgehog (IHH) ligands were detected in epididymal epithelial cells by confocal microscopy and found secreted in the extracellular space. Gene expression profiling preformed on ciliated epithelial cells indicated that 153 and 1052 genes were differentially expressed following treatment with the Hh agonist SAG or the Hh antagonist cyclopamine (Cyclo), respectively. Strikingly, gene ontology analysis indicated that genes associated with immune response were the most affected following Hh modulation. The contribution of epididymal PC to canonical Hh pathway transduction was validated by ciliobrevin D treatment, which induced a significant decrease in PC length and a reduction in the expression Hh signaling targets. Such findings bring us closer to a molecular understanding of the subtle immune balance observed in some epithelia, including the epididymis and the intestine, which are organs featuring both tolerance toward autoimmune spermatozoa (or commensal bacteria) and defense against pathogens.

Cell-lineage specificity of primary cilia during postnatal epididymal development.

STUDY QUESTION: Where are primary cilia (PC) organelles located during postnatal epididymal development? SUMMARY ANSWER: Our findings unveil the existence of PC sensory organelles in different epididymal cell types according to postnatal development stage. WHAT IS KNOWN ALREADY: Primary cilia are sensory organelles that orchestrate major signaling pathways during organ development and homeostasis. Epididymal PC have been detected in the horses, donkey and mules but their cell-lineage specificity has never been investigated in this organ. STUDY DESIGN, SIZE, DURATION: A longitudinal study was performed by examining tissue from n = 3 to n = 10 transgenic mice at different times of postnatal development. Tissues were fixed by intracardiac perfusion and the epididymides collected. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transmission electron microscopy and confocal microscopy/3D reconstruction were used on a double transgenic mouse model expressing endogenous fluorescence in PC and centrioles (Arl13b-mCherry/Centrin2-GFP). Several PC parameters (i.e. length, orientation relative to the lumen) were quantified by using an image-processing pipeline. Epididymal tissues and serum-free cultures of DC2 immortalized epididymal principal murine cell lines were used to identify primary ciliary signaling components. MAIN RESULTS AND THE ROLE OF CHANCE: We report here a constitutive localization of PC in peritubular myoid cells and a dynamic profiling in epithelial cells throughout postnatal epididymal development. While PC are present at the apical pole of the undifferentiated epithelial cells from birth to puberty, they are absent from the apical pole of the epithelium in adults, where they appear exclusively associated with cytokeratin 5-positive basal cells. We determined that PC from epididymal cells are associated with polycystin 1 (PC1), polycystin 2 (PC2), and Gli-3 Hedgehog signaling transcription factor. No inter-individual variability was observed within each age group. LIMITATIONS, REASONS FOR CAUTION: As our present study is descriptive and performed exclusively in the mouse, future functional studies will be required to unravel the contribution of these organelles in the control of reproductive functions. WIDER IMPLICATIONS OF THE FINDINGS: Acknowledging the important roles played by PC sensory organelles in organ homeostasis and development in humans, our work opens new avenues of research concerning the cellular control of epididymal functions, which are essential to male fertility. STUDY FUNDING/COMPETING INTEREST(S): Study funded by an NSERC operating grant to CB (RGPIN-2015-109194). No competing interest to declare.

Role of Dicer1-Dependent Factors in the Paracrine Regulation of Epididymal Gene Expression.

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.