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.

Intra and intercellular signals governing sperm maturation.

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.

The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans.

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.

The protein phosphatase with EF-hand domain 1 is a calmodulin-binding protein that interacts with proteins involved in sperm capacitation, binding to the zona pellucida, and motility.

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.

Study on the role of calmodulin in sperm function through the enrichment and identification of calmodulin-binding proteins in bovine ejaculated spermatozoa.

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.

Suboptimal extracellular pH values alter DNA damage response to induced double-strand breaks.

Conditions leading to unrepaired DNA double-stranded breaks are potent inducers of genetic instability. Systemic conditions may lead to fluctuation of hydrogen ions in the cellular microenvironment, and we show that small variations in extracellular pH, termed suboptimal pHe, can decrease the efficiency of DNA repair in the absence of intracellular pH variation. Recovery from bleomycin-induced DNA double-stranded breaks in fibroblasts proceeded less efficiently at suboptimal pHe values ranging from 7.2 to 6.9, as shown by the persistence of repair foci, reduction of H4K16 acetylation, and chromosomal instability, while senescence or apoptosis remained undetected. By allowing escape from these protective mechanisms, suboptimal pHe may therefore enhance the genotoxicity of double-stranded breaks, leading to genetic instability.

Loss of mesenchymal bone morphogenetic protein signaling leads to development of reactive stroma and initiation of the gastric neoplastic cascade.

Bmps are morphogens involved in various gastric cellular functions. Studies in genetically-modified mice have shown that Bmp disruption in gastric epithelial and stromal cell compartments leads to the development of tumorigenesis. Our studies have demonstrated that abrogation of gastric epithelial Bmp signaling alone was not sufficient to recapitulate the neoplastic features associated with total gastric loss of Bmp signaling. Thus, epithelial Bmp signaling does not appear to be a key player in gastric tumorigenesis initiation. These observations suggest a greater role for stromal Bmp signaling in gastric polyposis initiation. In order to identify the specific roles played by mesenchymal Bmp signaling in gastric homeostasis, we generated a mouse model with abrogation of Bmp signaling exclusively in the gastro-intestinal mesenchyme (Bmpr1a(ΔMES)). We were able to expose an unsuspected role for Bmp loss of signaling in leading normal gastric mesenchyme to adapt into reactive mesenchyme. An increase in the population of activated-fibroblasts, suggesting mesenchymal transdifferentiation, was observed in mutant stomach. Bmpr1a(ΔMES) stomachs exhibited spontaneous benign polyps with presence of both intestinal metaplasia and spasmolytic-polypeptide-expressing metaplasia as early as 90 days postnatal. These results support the novel concept that loss of mesenchymal Bmp signaling cascade acts as a trigger in gastric polyposis initiation.

Bmp signaling in colonic mesenchyme regulates stromal microenvironment and protects from polyposis initiation.

In the colon, myofibroblasts are primary contributors in the establishment of the microenvironment involved in tissue homeostasis. Alterations in myofibroblast functions lead to changes resulting in a toxic microenvironment nurturing tumorigenesis. Bone morphogenetic proteins (Bmps) are morphogens known to play key roles in adult gut homeostasis. Studies in genetically-modified mice have shown that Bmp disruption in all cell layers leads to the development of gut polyposis. In contrast, our studies showed that loss of Bmp exclusively in the gastrointestinal epithelium resulted in increased epithelial proliferation without polyposis initiation, thus suggesting a key role for mesenchymal Bmp signaling in polyposis initiation. In order to identify the role of mesenchymal Bmp signaling on the microenvironment and its impact on colonic mucosa, a mouse model was generated with suppression of Bmp signaling exclusively in myofibroblasts (Bmpr1aΔMES). Bmpr1aΔMES mice exhibited increased subepithelial proliferation with changes in cellular composition leading to the development of a primed stroma with modulation of extracellular matrix proteins, immune cells and cytokines as early as 90 days of age. This microenvironmental deregulation was associated with increased polyposis initiation at one year of age. These results are the first to demonstrate that mesenchymal Bmpr1a inactivation alone is sufficient to prompt an expansion of myofibroblasts leading to the development of a reactive mesenchyme that contributes to polyposis initiation in the colon. These findings support the novel concept that inhibition of Bmp signaling in mesenchymal cells surrounding the normal epithelium leads to important changes instructing a toxic microenvironment sufficient to induce colonic polyposis.