Mechanisms of release and signaling functions of ciliary vesicles

在哺乳动物的雄性生殖系统中，体细胞上皮细胞释放的胞外小泡通过分子货物的转移参与精子的成熟。这种独特的胞体与精子之间的通讯过程发生在附睾管上，它支持精子运动和受精能力的获得。尽管胞外小泡在维持雄性生殖功能方面发挥着重要作用，但它们的细胞来源、释放机制和各自的信号功能在很大程度上仍不清楚。初级纤毛是孤立的感觉触角，从细胞表面向上突出，形成一个富含信号分子的亚细胞室。来自这些细胞器的囊泡已被证明含有特定的生物活性分子子集。我们最近发现，初级纤毛丰富地存在于附睾管的上游，即传出小管中。在这个位置，它们释放细胞外小泡，表达特定的纤毛标记(如Ar13b和细胞色素P450氧化还原酶(POR))进入附睾液。基于附睾胞外纤毛小泡可能构成纤毛体细胞和成熟精子之间通讯的新途径的假设，我们提出了一个围绕三个主要目标的研究计划：目的1.表征男性生殖系统的纤毛小泡我们将利用Arl13b纤毛标记与m-Cherry标签融合的双转基因小鼠模型，通过专门的小颗粒流式细胞术方法(Collaboration E.Boilard，U.Laval)检测和分选原始纤毛来源的细胞外小泡。对m-樱桃阳性纤毛小泡的大小、蛋白质标志物和分子含量进行测定，并与附睾胞外m-樱桃阴性小泡进行比较。目的2.确定内皮细胞初级纤毛来源的囊泡的释放机制，这些囊泡是在切应力和鞭毛内转运(IFT)成分控制下质膜向外萌发形成的。我们将利用生物流体成像系统检测这两种因素对野生型和IFT88基因敲除小鼠传出小管上皮细胞原代培养的影响。目的3.评价它们对精子内容和功能的调节能力由于胞外囊泡通过传递生物活性分子参与细胞间的通讯，我们将结合体外共孵育试验和体内基因敲除小鼠模型的功能研究来评估它们在体细胞-精子通讯中的作用。NSERC的这项研究将揭开胞体到精子通讯系统中纤毛小泡的独特特征和功能，并可能识别控制精子成熟和男性生殖功能的新细胞机制。

In the mammalian male reproductive system, extracellular vesicles released by somatic epithelial cells participate to the maturation of spermatozoa through the transfer of molecular cargos. This unique process of soma-to-sperm communication occurs in the tubule attached to the testis, named the epididymis, and supports the acquisition of sperm motility and fertilizing abilities. Despite the important role played by extracellular vesicles to sustain male reproductive functions, their cellular source, mechanisms of release and respective signaling functions remain largely unknown. Primary cilia are solitary sensory antennae that protrude upward from the cell surface to form a subcellular compartment enriched in signaling molecules. Vesicles derived from these organelles have been shown to contain a specific subset of bioactive molecules. We recently unveiled that primary cilia are abundant in the tubules located upstream of the epididymis, i.e. in the efferent ductules. In this location, they release extracellular vesicles that express specific ciliary markers (e.g Arl13b and Cytochrome p450 oxidoreductase (POR)) into the epididymal fluid. With the hypothesis that ciliary extracellular vesicles from the epididymis may constitute news means of communication between ciliated somatic cells and the maturing spermatozoa, we propose a research program articulated around 3 majors aims: Aim 1. Characterize ciliary vesicles from the male reproductive system We will take advantage of a double transgenic mouse model, in which the Arl13b ciliary marker is fused with m-cherry tag, to detect and sort primary cilia-deriving extracellular vesicles by dedicated small particle flow-cytometry approaches (collaboration E. Boilard, U.Laval). The size, protein markers and molecular content of m-cherry positive ciliary vesicles will be determined and compared with m-cherry negative extracellular vesicles from the epididymis. Aim 2. Determine their mechanisms of release Vesicles that derive from the primary cilium of endothelial cells are formed by outward budding of the plasma membrane under the control of shear stress and intraflagellar transport (IFT) components. We will test the contribution of these two factors on epithelial primary cell cultures isolated from the efferent ductules of wild type and IFT88 knock-out mice by using biofludic imaging system. Aim 3. Assess their ability to modulate sperm content and function Since extracellular vesicles participate to intercellular communication via the transfer of bioactive molecules, we will assess their role in somatic cells-spermatozoa communication by combining in vitro co-incubation assays with in vivo functional studies on knock-out mouse models. This NSERC research will unravel the unique features and functions of ciliary vesicles in the soma-to-sperm communication system and may identify new cellular mechanisms that control sperm maturation and male reproductive functions.