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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.

After their production in the testis, spermatozoa do not have the capacity to move progressively and are unable to fertilise an oocyte. They sequentially acquire these abilities following their maturation in the epididymis and their capacitation/hyperactivation in the female reproductive system. As gene transcription is silenced in spermatozoa, extracellular factors released from the epididymal epithelium and from secretory glands allow spermatozoa to acquire bioactive molecules and to undergo intrinsic modifications. These modifications include epigenetic changes and post-translational modifications of endogenous proteins, which are important processes in sperm maturation. This article emphasises the roles played by extracellular factors secreted by the epididymis and accessory glands in the control of sperm intercellular signallings and fertilising abilities.

Spermatozoa are highly specialized cells whose fertilizing and motility functions highly depend on intracellular Ca2+ -mediated events and protein posttranslational modifications like phosphorylation. Our group previously identified PPEF1, the Ser/Thr phosphatase with EF-hand domain 1, among calmodulin-affinity pulled down sperm proteins. As the mammalian ortholog of the Drosophila phosphatase rdgC that dephosphorylates rhodopsin, PPEF1 has been studied mostly in the retina. The presence and importance of this Ca2+ /calmodulin-binding protein phosphatase has not been studied in sperm or testicular functions despite its high expression level. In this study, we show that PPEF1 is present in testicular germ cells, and in mouse, human and bull spermatozoa where it is localized predominantly in the neck and acrosome areas. Different transcript variants encoding four predicted isoforms were detected by reverse transcription polymerase chain reaction in bull testis, spermatocytes and spermatids. Phosphatase activity of immunoprecipitated sperm PPEF1 was detected using the substrate pNPP and analysis of the coimmunoprecipitated proteins reveal an enrichment in the biological processes of sperm capacitation, binding to the zona pellucida and motility. Although this is the first demonstration of the presence of PPEF1 in sperm and testicular germ cells, its involvement in sperm fertilizing ability and motility, and the mechanisms regulating its activity remain to be further investigated.

Calmodulin is a small, highly conserved acidic protein present at high levels in spermatozoa that mediates numerous intracellular Ca2+ -dependent events. Sperm motility and fertilizing ability results from an array of biochemical pathways under Ca2+ control, in which the importance of calmodulin is not fully understood. The role of calmodulin in sperm function has been mostly assessed using antagonists. Nevertheless, few known calmodulin-regulated enzymes have been described in spermatozoa regarding their involvement in sperm function. To further understand the role of this important Ca2+ mediator in spermatozoa, different studies were also undertaken to investigate and to identify sperm calmodulin-binding proteins and determine their localization and subcellular distribution as an attempt to elucidate the role of this important Ca2+ mediator. In the present study, sperm calmodulin-binding proteins were identified by mass spectrometry after Ca2+ -dependent biotinylated-calmodulin binding on sperm head proteins subjected to 2D electrophoresis and transferred on a polyvinylidene difluoride membrane. Calmodulin binding protein identification was also done on detergent extracted whole sperm proteins pulled down in a Ca2+ -dependent manner by calmodulin-conjugated sepharose beads. In this latter group, 300 proteins were identified in at least two experiments out of three, and those identified in the three independent experiments were analyzed for overrepresented biological processes using the Bos taurus Gene Ontology database. Proteins with known function in reproductive processes, fertilization, sperm-egg recognition, sperm binding to the zona pellucida, regulation of sperm capacitation, and sperm motility were identified and further emphasize the importance of calmodulin in sperm function.

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3’UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo., Author summary MicroRNAs play an essential contribution among the many mechanisms used by cells to maintain a proper gene expression program. These endogenous small RNAs regulate gene expression by binding to target mRNAs and blocking their translation into functional proteins. To achieve this, microRNAs rely on Argonaute and other proteins that form a complex called the miRISC. While research on microRNAs has brought a lot of interest over the last years, it is still unclear what modulators affect the activity of the miRISC. Our study identified proteins implicated in intracellular trafficking as important factors for miRISC function. We showed that the intracellular location of the Argonaute protein needs to be tightly regulated to assure proper microRNA-mediated mRNA repression in animals.